{"id":907,"date":"2021-08-11T09:34:05","date_gmt":"2021-08-11T09:34:05","guid":{"rendered":"https:\/\/blog.praxilabs.com\/?p=907"},"modified":"2025-08-22T23:08:40","modified_gmt":"2025-08-22T23:08:40","slug":"how-to-analyze-western-blot-data","status":"publish","type":"post","link":"https:\/\/praxilabs.com\/en\/blog\/2021\/08\/11\/how-to-analyze-western-blot-data\/","title":{"rendered":"How to Analyze Western Blot Data"},"content":{"rendered":"<p><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><span style=\"font-weight: 400;\">Western blot is an indispensable mechanism in the modern biomedical research laboratory, as well as in laboratories doing research in other areas. It is considered as an analytical technique used mainly in molecular biology and <\/span><a href=\"https:\/\/en.wikipedia.org\/wiki\/Immunogenetics\" target=\"_blank\" rel=\"noopener\"><span style=\"font-weight: 400;\"><span style=\"color: #0000ff;\">immunogenetics<\/span><\/span><\/a><span style=\"font-weight: 400;\"> where antibodies are used to specifically detect their antigen.<\/span><\/span><\/p>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Before discussing how to analyze western blot data, you can know more about the concept and processes of western blot from our article <span style=\"color: #0000ff;\">&#8220;<a style=\"color: #0000ff; text-decoration: underline;\" href=\"https:\/\/praxilabs.com\/en\/blog\/2020\/05\/29\/western-blot-concept\/\">Western Blot: Concept, How to Use It, and Its Applications.<\/a>&#8220;<\/span><\/span><\/p>\n<p style=\"text-align: center;\"><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><strong><a class=\"maxbutton-3 maxbutton\" href=\"https:\/\/praxilabs.com\/en\/3d-simulations\/western-blot-biology-virtual-lab-simulation\"><span class='mb-text'>Try Western Blot in Praxilabs Virtual Labs<\/span><\/a><\/strong><\/span><\/p>\n<p><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><img loading=\"lazy\" decoding=\"async\" class=\"size-full wp-image-909 aligncenter\" src=\"https:\/\/praxilabs.com\/en\/blog\/wp-content\/uploads\/2021\/08\/image.jpg\" alt=\"how to analyze western blot data\" width=\"673\" height=\"448\" srcset=\"https:\/\/praxilabs.com\/en\/blog\/wp-content\/uploads\/2021\/08\/image.jpg 673w, https:\/\/praxilabs.com\/en\/blog\/wp-content\/uploads\/2021\/08\/image-300x200.jpg 300w, https:\/\/praxilabs.com\/en\/blog\/wp-content\/uploads\/2021\/08\/image-310x205.jpg 310w\" sizes=\"auto, (max-width: 673px) 100vw, 673px\" \/><\/span><\/p>\n<div id=\"ez-toc-container\" class=\"ez-toc-v2_0_84 counter-hierarchy ez-toc-counter ez-toc-light-blue ez-toc-container-direction\">\r\n<div class=\"ez-toc-title-container\">\r\n<p class=\"ez-toc-title\" style=\"cursor:inherit\">Table of Contents<\/p>\r\n<span class=\"ez-toc-title-toggle\"><\/span><\/div>\r\n<nav><ul class='ez-toc-list ez-toc-list-level-1 ' ><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-1\" href=\"https:\/\/praxilabs.com\/en\/blog\/2021\/08\/11\/how-to-analyze-western-blot-data\/#Western_Blot\" >Western Blot<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-2\" href=\"https:\/\/praxilabs.com\/en\/blog\/2021\/08\/11\/how-to-analyze-western-blot-data\/#Western_Blotting_Steps\" >Western Blotting Steps<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-3\" href=\"https:\/\/praxilabs.com\/en\/blog\/2021\/08\/11\/how-to-analyze-western-blot-data\/#How_to_Analyze_Western_Blot_Data\" >How to Analyze Western Blot Data?<\/a><ul class='ez-toc-list-level-3' ><li class='ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-4\" href=\"https:\/\/praxilabs.com\/en\/blog\/2021\/08\/11\/how-to-analyze-western-blot-data\/#The_Steps_for_Western_Blot_Quantification\" >The Steps for Western Blot Quantification\u00a0<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-5\" href=\"https:\/\/praxilabs.com\/en\/blog\/2021\/08\/11\/how-to-analyze-western-blot-data\/#Example_of_Western_Blot_Quantification_Graph\" >Example of Western Blot Quantification Graph<\/a><\/li><\/ul><\/li><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-6\" href=\"https:\/\/praxilabs.com\/en\/blog\/2021\/08\/11\/how-to-analyze-western-blot-data\/#Western_Blotting_Results_and_Discussion\" >Western Blotting Results and Discussion<\/a><ul class='ez-toc-list-level-3' ><li class='ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-7\" href=\"https:\/\/praxilabs.com\/en\/blog\/2021\/08\/11\/how-to-analyze-western-blot-data\/#How_Would_You_Describe_Western_Blot_Data\" >How Would You Describe Western Blot Data?<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-8\" href=\"https:\/\/praxilabs.com\/en\/blog\/2021\/08\/11\/how-to-analyze-western-blot-data\/#Typical_Western_Transfer_and_Development_Protocol\" >Typical Western Transfer and Development Protocol<\/a><ul class='ez-toc-list-level-4' ><li class='ez-toc-heading-level-4'><a class=\"ez-toc-link ez-toc-heading-9\" href=\"https:\/\/praxilabs.com\/en\/blog\/2021\/08\/11\/how-to-analyze-western-blot-data\/#_Transfer_Protocol\" >\u00a0Transfer Protocol:<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-4'><a class=\"ez-toc-link ez-toc-heading-10\" href=\"https:\/\/praxilabs.com\/en\/blog\/2021\/08\/11\/how-to-analyze-western-blot-data\/#Development_Immunostaining_Protocol\" >Development (Immunostaining) Protocol:<\/a><\/li><\/ul><\/li><\/ul><\/li><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-11\" href=\"https:\/\/praxilabs.com\/en\/blog\/2021\/08\/11\/how-to-analyze-western-blot-data\/#Western_Blotting_Common_Questions\" >Western Blotting Common Questions<\/a><ul class='ez-toc-list-level-3' ><li class='ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-12\" href=\"https:\/\/praxilabs.com\/en\/blog\/2021\/08\/11\/how-to-analyze-western-blot-data\/#What_is_the_difference_between_Elisa_and_Western_Blot\" >What is the difference between Elisa and Western Blot?<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-13\" href=\"https:\/\/praxilabs.com\/en\/blog\/2021\/08\/11\/how-to-analyze-western-blot-data\/#What_Does_a_Western_Blot_Tell_You\" >What Does a Western Blot Tell You?<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-14\" href=\"https:\/\/praxilabs.com\/en\/blog\/2021\/08\/11\/how-to-analyze-western-blot-data\/#How_Long_Does_Western_Blot_Test_Take\" >How Long Does Western Blot Test Take?<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-15\" href=\"https:\/\/praxilabs.com\/en\/blog\/2021\/08\/11\/how-to-analyze-western-blot-data\/#What_Is_the_Purpose_of_the_Transfer_in_Western_Blot_protocol\" >What Is the Purpose of the Transfer in Western Blot protocol?<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-16\" href=\"https:\/\/praxilabs.com\/en\/blog\/2021\/08\/11\/how-to-analyze-western-blot-data\/#Can_Western_Blot_Be_Quantitative\" >Can Western Blot Be Quantitative?<\/a><\/li><\/ul><\/li><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-17\" href=\"https:\/\/praxilabs.com\/en\/blog\/2021\/08\/11\/how-to-analyze-western-blot-data\/#Western_Blot_PraxiLabs_Virtual_Lab\" >Western Blot\u00a0 PraxiLabs Virtual Lab<\/a><\/li><\/ul><\/nav><\/div>\r\n<h2><span class=\"ez-toc-section\" id=\"Western_Blot\"><\/span><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><b>Western Blot<\/b><\/span><span class=\"ez-toc-section-end\"><\/span><\/h2>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">The transfer of proteins or nucleic acids to microporous membranes is referred to as \u201cblotting\u201d and this term encompasses both \u201cspotting\u201d (manual sample deposition) and transfer from planar gels.<\/span><\/p>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Proteins that are resolved on sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) gels are typically transferred to adsorbent membrane supports under the influence of an electric current in a procedure that is known as Western blotting (WB) or protein blotting.<\/span><\/p>\n<h2><span class=\"ez-toc-section\" id=\"Western_Blotting_Steps\"><\/span><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><b>Western Blotting Steps<\/b><\/span><span class=\"ez-toc-section-end\"><\/span><\/h2>\n<p><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><span style=\"font-size: 14pt;\"><span style=\"font-weight: 400;\">1-\u00a0 <\/span><span style=\"font-weight: 400;\">The first step in a western blotting is preparing samples: <\/span><\/span><span style=\"font-size: 14pt;\"><span style=\"font-weight: 400;\">The samples are prepared and loaded onto a gel. Your sample could be tissue, cells, or another solution that you want to extract and analyze its protein.<\/span><\/span><span style=\"font-size: 14pt;\"><span style=\"font-weight: 400;\">\u00a0<\/span><\/span><\/span><\/p>\n<p><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><span style=\"font-weight: 400;\">2-\u00a0 <\/span><span style=\"font-weight: 400;\">Electrophoretic separation of proteins: The procedure is to separate the macromolecules in a sample using gel electrophoresis. The separated molecules are transferred or blotted onto a second matrix, generally a nitrocellulose or polyvinylidene difluoride (PVDF) membrane.<\/span><\/span><\/p>\n<p><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><span style=\"font-weight: 400;\">\u00a0 3- <\/span><span style=\"font-weight: 400;\">Blocking nonspecific sites:<\/span> <span style=\"font-weight: 400;\">The membrane is blocked to prevent any nonspecific binding of <span style=\"color: #0000ff;\"><a style=\"color: #0000ff; text-decoration: underline;\" href=\"https:\/\/en.wikipedia.org\/wiki\/Antibody\" target=\"_blank\" rel=\"noopener\">antibodies<\/a><\/span> to the surface of the membrane.<\/span><\/span><\/p>\n<p><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><span style=\"font-weight: 400;\">\u00a0 4- <\/span><span style=\"font-weight: 400;\">The transferred protein is then probed with a combination of antibodies: one antibody specific to the protein of interest (primary antibody) and another antibody specific to the host species of the primary antibody (secondary antibody). Often the secondary antibody is complexed with an enzyme, which when combined with an appropriate substrate will produce a detectable signal.<\/span><\/span><\/p>\n<p><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><span style=\"font-weight: 400;\">\u00a0 5- <\/span><span style=\"font-weight: 400;\">Detection Methods:<\/span> <span style=\"font-weight: 400;\">In the detection step, the protein-antibody-antibody complex is detected on the membrane. There are various detection systems, based on <span style=\"color: #000000;\">chemiluminescence<\/span>, chemifluorescence, fluorescence, chromogenic, or radioisotopic detection.<\/span><\/span><\/p>\n<p><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><span style=\"font-weight: 400;\">6-\u00a0 <\/span><span style=\"font-weight: 400;\">Imaging,<\/span> <span style=\"font-weight: 400;\">which is the last step in the Western blotting workflow before data analysis, is image capture. You can perform the capturing step using a film, a CCD camera, or a scanner to collect the emitted light of the detection process.<\/span><\/span><\/p>\n<p><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><span style=\"font-weight: 400;\">\u00a07- <\/span><span style=\"font-weight: 400;\">Analysis: The detected signals, using either X-ray film, scanners, or a charge-coupled device CCD camera, cause one or more visible protein bands on the membrane image.\u00a0<\/span><\/span><\/p>\n<p style=\"text-align: center;\"><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><strong><a class=\"maxbutton-3 maxbutton\" href=\"https:\/\/praxilabs.com\/en\/pricing\"><span class='mb-text'>Try Western Blot Now For FREE!<\/span><\/a><\/strong><\/span><\/p>\n<h2><span class=\"ez-toc-section\" id=\"How_to_Analyze_Western_Blot_Data\"><\/span><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><b>How to Analyze Western Blot Data?<\/b><\/span><span class=\"ez-toc-section-end\"><\/span><\/h2>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">The data produced with a Western blot is usually quite easy to interpret. In the majority of cases, bands corresponding to the target protein will become visible upon treatment of the blot with substrate. Their identity is confirmed by comparison to molecular weight markers (for size) and a positive control (size and signal).<\/span><\/p>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">In some cases the data may be more complex, showing unexpected sizes, multiple bands, or alteration in bands following a particular treatment.<\/span><\/p>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">To estimate the molecular weight of the protein you can make a comparison with marker proteins and the amount of protein can be determined as this is related to band intensity (within the limits of the detection system).\u00a0<\/span><\/p>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">In most applications, it is enough to confirm protein presence and roughly estimate the amount. However, other applications demand a quantitative analysis that defines protein levels in either relative or absolute terms.<\/span><\/p>\n<h3><span class=\"ez-toc-section\" id=\"The_Steps_for_Western_Blot_Quantification\"><\/span><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><b>The Steps for Western Blot Quantification\u00a0<\/b><\/span><span class=\"ez-toc-section-end\"><\/span><\/h3>\n<p><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><strong><span style=\"font-size: 14pt;\">(Quantitative Western Blot protocol)<\/span><\/strong><\/span><\/p>\n<ol>\n<li><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><strong><span style=\"font-size: 14pt;\"> Find the Linear Range<\/span><\/strong><\/span><\/li>\n<\/ol>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">For quantitative analysis of an image you must ensure your image was captured in a manner sensitive enough to detect change, in what we call the \u201clinear range.\u201d If you are not working within the linear range, e.g., if your detector or film can no longer absorb photons, it is saturated and you have hit your limit of detection, you are losing data. You don\u2019t want this.<\/span><\/p>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">\u00a0Many digital capturing systems come with software designed to detect saturation and automatically correct the exposure thereby ensuring your data analysis is quantitative. So take the time to formally review your software and see if this is your case. However, film can easily become saturated.<\/span><\/p>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">To prevent saturation on film, you must empirically determine your linear range. To do this you need to serially dilute a known amount of your protein lysate, perform your Western, and plot the quantitated density of these Western blot bands against the amount you know you loaded.<\/span><\/p>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"> You should then find a linear line indicating where data is captured quantitatively. This is where you want to work. To fix any saturation problems, you can try loading less total protein and\u00a0 less primary antibody dilution, try a new antibody, or reduce the film exposure length. You need to go through this process for each antibody separately.<\/span><\/p>\n<ol start=\"2\">\n<li><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><strong><span style=\"font-size: 14pt;\"> Subtract Background<\/span><\/strong><\/span><\/li>\n<\/ol>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Sadly, most Western blots and image captures are infiltrated with random imperfections. For example, the left side of the blot may be a little darker (higher background) or your less abundant band might have more background or an annoying dark scratch.<\/span><\/p>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"> These differences can cause inconsistencies in your results. Many software packages can calculate the background around your band of interest, using some variation of the \u201crolling ball\u201d method (again, take time to understand your software). The background should be subtracted from both your bands of interest and the bands you are normalizing to. Perfection here is challenging; just do your best and let statistics estimate the real answer when you are all done.<\/span><\/p>\n<ol start=\"3\">\n<li><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><strong><span style=\"font-size: 14pt;\"> Normalize<\/span><\/strong><\/span><\/li>\n<\/ol>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Variability happens in Western blotting. You may have transferred unevenly, loaded too little in one lane, or maybe no one believes your data and they just want to see that you controlled everything. This is why normalization exists. To control for variability we often normalize to another band in the blot, typically an abundant protein that we don\u2019t expect to change in our experiment.<\/span><\/p>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"> These control proteins are often produced from a \u201chousekeeping gene\u201d such as actin, beta-tubulin or a chaperone protein like Hsp70. However, as many of us have discovered, these proteins can unexpectedly change in our experimental conditions. And, due to their high abundance, they can also be challenging to acquire in the linear range. Sometimes choosing a random background band that doesn\u2019t change is the best choice.<\/span><\/p>\n<p><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><strong><span style=\"font-size: 14pt;\">Steps to Normalize the Protein Band of Interest:<\/span><\/strong><\/span><\/p>\n<p><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><strong>(Western Blot Normalization Calculation)<\/strong><\/span><\/p>\n<p><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><b>Step 1:<\/b><span style=\"font-weight: 400;\"> Determine the background-subtracted densities of your protein of interest (PI) and the normalizing control (NC).<\/span><\/span><\/p>\n<p><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><b>Step 2:<\/b><span style=\"font-weight: 400;\"> Identify the NC that has the highest density value.<\/span><\/span><\/p>\n<p><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><b>Step 3:<\/b><span style=\"font-weight: 400;\"> Divide all the NC values by the highest NC density value to get a relative NC value. If you do this correctly the highest density value will be 1, and the others a fraction of it (e.g., 0.97).<\/span><\/span><\/p>\n<p><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><b>Step 4:<\/b><span style=\"font-weight: 400;\"> Divide all of your PI values by the relative NC values in their respective lanes.<\/span><\/span><\/p>\n<ol start=\"4\">\n<li><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><strong> Graphs and Stats (Western Blot Statistical Analysis)<\/strong><\/span><\/li>\n<\/ol>\n<p><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><span style=\"font-weight: 400; font-size: 14pt;\">Once you have obtained normalized values you are ready to crunch the numbers and view your results. Typically for quantitative experiments you should perform each condition in triplicate (preferably on the same blot)<\/span><span style=\"font-weight: 400; font-size: 14pt;\">.<\/span><\/span><\/p>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"> After you have determined your normalized values for each replicate, you can determine averages, p-values, fold changes, and\/or graph results. Then doing the entire experiment three independent times ensures that your results aren\u2019t a fluke and are indeed repeatable.\u00a0<\/span><\/p>\n<p style=\"text-align: center;\"><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><strong><a class=\"maxbutton-3 maxbutton\" href=\"https:\/\/praxilabs.com\/en\/pricing\"><span class='mb-text'>Get Started Praxilabs For FREE<\/span><\/a><\/strong><\/span><\/p>\n<h3><span class=\"ez-toc-section\" id=\"Example_of_Western_Blot_Quantification_Graph\"><\/span><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\">Example of Western Blot Quantification Graph<\/span><span class=\"ez-toc-section-end\"><\/span><\/h3>\n<p><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><img loading=\"lazy\" decoding=\"async\" class=\"alignnone size-full wp-image-910\" src=\"https:\/\/praxilabs.com\/en\/blog\/wp-content\/uploads\/2021\/08\/A-Representative-Western-blot-and-quantification-graphs-of-basal-protein-expression-of.png\" alt=\"Example of Western Blot Quantification Graph\" width=\"850\" height=\"1427\" srcset=\"https:\/\/praxilabs.com\/en\/blog\/wp-content\/uploads\/2021\/08\/A-Representative-Western-blot-and-quantification-graphs-of-basal-protein-expression-of.png 850w, https:\/\/praxilabs.com\/en\/blog\/wp-content\/uploads\/2021\/08\/A-Representative-Western-blot-and-quantification-graphs-of-basal-protein-expression-of-179x300.png 179w, https:\/\/praxilabs.com\/en\/blog\/wp-content\/uploads\/2021\/08\/A-Representative-Western-blot-and-quantification-graphs-of-basal-protein-expression-of-610x1024.png 610w, https:\/\/praxilabs.com\/en\/blog\/wp-content\/uploads\/2021\/08\/A-Representative-Western-blot-and-quantification-graphs-of-basal-protein-expression-of-768x1289.png 768w\" sizes=\"auto, (max-width: 850px) 100vw, 850px\" \/><\/span><\/p>\n<h2><span class=\"ez-toc-section\" id=\"Western_Blotting_Results_and_Discussion\"><\/span><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\">Western Blotting Results and Discussion<\/span><span class=\"ez-toc-section-end\"><\/span><\/h2>\n<h3><span class=\"ez-toc-section\" id=\"How_Would_You_Describe_Western_Blot_Data\"><\/span><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\" data-sheets-value=\"{&quot;1&quot;:2,&quot;2&quot;:&quot;How would you describe Western blot data?&quot;}\" data-sheets-userformat=\"{&quot;2&quot;:1325763,&quot;3&quot;:{&quot;1&quot;:0},&quot;4&quot;:{&quot;1&quot;:2,&quot;2&quot;:16776960},&quot;9&quot;:1,&quot;10&quot;:0,&quot;12&quot;:0,&quot;14&quot;:{&quot;1&quot;:2,&quot;2&quot;:0},&quot;15&quot;:&quot;arial, sans-serif&quot;,&quot;16&quot;:12,&quot;21&quot;:0,&quot;23&quot;:2}\">How Would You Describe Western Blot Data?<\/span><span class=\"ez-toc-section-end\"><\/span><\/h3>\n<p><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><img loading=\"lazy\" decoding=\"async\" class=\"size-full wp-image-911 aligncenter\" src=\"https:\/\/praxilabs.com\/en\/blog\/wp-content\/uploads\/2021\/08\/image-1.jpg\" alt=\"How to Read Western Blot Results\" width=\"673\" height=\"448\" srcset=\"https:\/\/praxilabs.com\/en\/blog\/wp-content\/uploads\/2021\/08\/image-1.jpg 673w, https:\/\/praxilabs.com\/en\/blog\/wp-content\/uploads\/2021\/08\/image-1-300x200.jpg 300w, https:\/\/praxilabs.com\/en\/blog\/wp-content\/uploads\/2021\/08\/image-1-310x205.jpg 310w\" sizes=\"auto, (max-width: 673px) 100vw, 673px\" \/><\/span><\/p>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"> To know how to analyze western blot data, Look for the sizes of the bands. These will be represented by a number, either followed by &#8220;kDa&#8221; or preceded by &#8220;p.&#8221; This is the size of the protein which has been detected and is the scale on which the proteins are separated in a Western blot. It is these differently numbered bands that represent different proteins and will determine a positive result or not.<\/span><\/p>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Determine which bands have a positive result and what this may mean. For example, with the test for Lyme disease, there are several different bands which may produce a positive result, and any one of these will mean a positive result.<\/span><\/p>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Other tests may simply produce non-significant bands. These may not be reported, but ensure it is clear which are the important bands. Specific bands which show a positive result means a positive result for the infection that was being tested for.<\/span><\/p>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Discuss the results and any concerns or questions with a clinician. A positive result may require some treatment, and the consequences of this should also be discussed with the clinician who ordered the Western blot diagnostic test.<\/span><\/p>\n<h3><span class=\"ez-toc-section\" id=\"Typical_Western_Transfer_and_Development_Protocol\"><\/span><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><b>Typical Western Transfer and Development Protocol<\/b><\/span><span class=\"ez-toc-section-end\"><\/span><\/h3>\n<p><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><strong><span style=\"font-size: 14pt;\">Materials Needed:<\/span><\/strong><\/span><\/p>\n<ul>\n<li style=\"font-weight: 400;\" aria-level=\"1\"><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">SDS-PAGE apparatus and accessories<\/span><\/li>\n<li style=\"font-weight: 400;\" aria-level=\"1\"><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Submerged Western Transfer Cassette and accessories<\/span><\/li>\n<li style=\"font-weight: 400;\" aria-level=\"1\"><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">A nitrocellulose membrane, approximately the size of the gel, presoaked in Western Transfer Buffer* for five minutes. (Note: The membrane should be handled with gloves and clean forceps to avoid contamination with extraneous proteins.)<\/span><\/li>\n<li style=\"font-weight: 400;\" aria-level=\"1\"><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><span style=\"font-weight: 400;\">*Western Transfer Buffer (1\/2 x TOWBIN)<\/span><span style=\"font-weight: 400;\"><br \/>\n<\/span><span style=\"font-weight: 400;\"> Tris Base&#8230;&#8230;&#8230;&#8230;..1.45g (12mM final concentration)<\/span><span style=\"font-weight: 400;\"><br \/>\n<\/span><span style=\"font-weight: 400;\"> Glycine&#8230;&#8230;&#8230;&#8230;&#8230;&#8230;7.2g (96mM final concentration)<\/span><span style=\"font-weight: 400;\"><br \/>\n<\/span><span style=\"font-weight: 400;\"> Methanol&#8230;&#8230;&#8230;&#8230;..200mL (20% final volume)<\/span><span style=\"font-weight: 400;\"><br \/>\n<\/span><span style=\"font-weight: 400;\"> diH2O&#8230;&#8230;&#8230;&#8230;&#8230;&#8230;..add to 1.0L final volume<\/span><\/span><\/li>\n<li style=\"font-weight: 400;\" aria-level=\"1\"><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Antigen specific probing antibody (PrimaryAntibody)<\/span><\/li>\n<li style=\"font-weight: 400;\" aria-level=\"1\"><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Secondary antibody (Donkey-anti-probing antibody species conjugated to Alkaline Phosphatase)<\/span><\/li>\n<li style=\"font-weight: 400;\" aria-level=\"1\"><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Commercially available alkaline phosphatase conjugate substrate kit<\/span><\/li>\n<\/ul>\n<h4><span class=\"ez-toc-section\" id=\"_Transfer_Protocol\"><\/span><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><span style=\"font-weight: 400;\">\u00a0<\/span><strong>Transfer Protocol<\/strong>:<\/span><span class=\"ez-toc-section-end\"><\/span><\/h4>\n<ol>\n<li><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Load the protein sample onto a 4-20% Tris-Glycine polyacrylamide gel and run until desired resolution is achieved. (The electrophoresis can be followed by using pre-stained molecular weight markers).<\/span><\/li>\n<li><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Set up the Submerged Western Transfer Cassette.<\/span><\/li>\n<li><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Submerge the open Transfer Cassette cathode plate onto the tray pre-filled with Western Transfer Buffer.<\/span><\/li>\n<li><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Place a sponge support pad onto the cassette and remove the air bubbles by gently rolling a Pasteur pipette over the pad.<\/span><\/li>\n<li><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Place a piece of blotting paper onto the sponge support pad.<\/span><\/li>\n<li><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Remove the gel from the electrophoresis plates, cut off approximately the bottom 3mm of the gel so that the membrane can be laid flat against the gel, and place the gel over the blotting paper. Expel air bubbles as before.<\/span><\/li>\n<li><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Carefully place the pre-soaked nitrocellulose membrane onto the gel and expel air bubbles. Ensure that the membrane remains directly over the gel before proceeding.<\/span><\/li>\n<li><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><span style=\"font-weight: 400;\"> \u00a0 <\/span> <span style=\"font-weight: 400;\">Place a second piece of blotting paper onto the nitrocellulose membrane and remove air bubbles.<\/span><\/span><\/li>\n<li><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Place a sponge support pad onto the second piece of blotting paper and remove air bubbles.<\/span><\/li>\n<li><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Gently close the cassette by placing the anode plate over the exposed pad.<\/span><\/li>\n<li><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Carefully place the assembled cassette into the transfer tank containing Western Transfer Buffer up to the &#8220;pre-fill&#8221; level and adjust the buffer level, as needed, after the addition of the cassette.<\/span><\/li>\n<li><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><span style=\"font-weight: 400; font-size: 14pt;\"><span style=\"font-weight: 400; font-size: 14pt;\">Connect the assembled apparatus to an electrophoresis power supply and run for approximately 1.5 hours at a constant current of 400mA.<\/span><\/span><\/span><\/li>\n<\/ol>\n<h4><span class=\"ez-toc-section\" id=\"Development_Immunostaining_Protocol\"><\/span><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><strong>Development (Immunostaining) Protocol<\/strong>:<\/span><span class=\"ez-toc-section-end\"><\/span><\/h4>\n<ol>\n<li><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><span style=\"font-weight: 400;\">After the transfer is complete, incubate the membrane in a blocking solution (3% Nonfat Dry Milk in diH<\/span><span style=\"font-weight: 400;\">2<\/span><span style=\"font-weight: 400;\">O) for 30 minutes with gentle agitation on an orbital shaker.<\/span><\/span><\/li>\n<li><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Wash the membrane three times with TBST (TBS, pH 7.2 with 0.1% TWEEN-20) in a clean tray on an orbital shaker; each wash lasting 5-10 minutes.<\/span><\/li>\n<li><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Dilute the probing (primary) antibody in TBST to a volume of 50ml (approximate final concentration of 0.20\u00b5g\/ml) and incubate the membrane in the antibody solution for one to four hours at room temperature. (The optimum incubation time depends on the antibody\/antigen binding affinity and must be pre-determined for each antibody.)<\/span><\/li>\n<li><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Wash the membrane three times as in step #2.<\/span><\/li>\n<li><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Dilute the secondary antibody in TBST according to the manufacturer&#8217;s specification. Incubate the membrane in a clean tray containing 50ml of diluted secondary antibody for one hour at room temperature on an orbital shaker.<\/span><\/li>\n<li><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Wash the membrane three times as in step #2.<\/span><\/li>\n<li><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Color development requires the use of a commercially available (e.g. Bio-Rad or Sigma) alkaline phosphatase conjugate substrate kit. Follow the manufacturer&#8217;s instructions.<\/span><\/li>\n<li><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><span style=\"font-weight: 400;\">After the bands become clearly visible, stop the color by placing the membrane in a tray containing diH<\/span><span style=\"font-weight: 400;\">2<\/span><span style=\"font-weight: 400;\">O for at least ten minutes.<\/span><\/span><\/li>\n<\/ol>\n<p><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><img loading=\"lazy\" decoding=\"async\" class=\"size-full wp-image-912 aligncenter\" src=\"https:\/\/praxilabs.com\/en\/blog\/wp-content\/uploads\/2021\/08\/Smart-Answers-to-the-21-Most-Common-Interview-Questions-in-2021-2.png\" alt=\"Western Blotting Common Questions\" width=\"850\" height=\"420\" srcset=\"https:\/\/praxilabs.com\/en\/blog\/wp-content\/uploads\/2021\/08\/Smart-Answers-to-the-21-Most-Common-Interview-Questions-in-2021-2.png 850w, https:\/\/praxilabs.com\/en\/blog\/wp-content\/uploads\/2021\/08\/Smart-Answers-to-the-21-Most-Common-Interview-Questions-in-2021-2-300x148.png 300w, https:\/\/praxilabs.com\/en\/blog\/wp-content\/uploads\/2021\/08\/Smart-Answers-to-the-21-Most-Common-Interview-Questions-in-2021-2-768x379.png 768w\" sizes=\"auto, (max-width: 850px) 100vw, 850px\" \/><\/span><\/p>\n<h2><span class=\"ez-toc-section\" id=\"Western_Blotting_Common_Questions\"><\/span><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><b>Western Blotting Common Questions<\/b><\/span><span class=\"ez-toc-section-end\"><\/span><\/h2>\n<h3><span class=\"ez-toc-section\" id=\"What_is_the_difference_between_Elisa_and_Western_Blot\"><\/span><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><b>What is the difference between Elisa and Western Blot?<\/b><\/span><span class=\"ez-toc-section-end\"><\/span><\/h3>\n<table style=\"width: 99.5785%; height: 595px;\">\n<tbody>\n<tr style=\"height: 60px;\">\n<td style=\"width: 47.4197%; height: 60px;\">\n<p style=\"text-align: center;\"><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><b>Elisa<\/b><\/span><\/p>\n<\/td>\n<td style=\"width: 51.7373%; height: 60px;\">\n<p style=\"text-align: center;\"><span style=\"font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"><b>Western Blot<\/b><\/span><\/p>\n<\/td>\n<\/tr>\n<tr style=\"height: 172px;\">\n<td style=\"width: 47.4197%; height: 172px;\"><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">The ELISA test uses enzymes or antibodies attached to a solid surface to create the test surface. A sample is then added to the test surface. Antibodies or enzymes reacting or attaching to proteins indicates a positive result.<\/span><\/td>\n<td style=\"width: 51.7373%; height: 172px;\"><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">The Western blot test is performed after the gel-electrophoresis. The separated proteins are transferred (or blotted) onto nitrocellulose or nylon membranes and identified by specific antibodies that are tagged by a secondary protein.<\/span><\/td>\n<\/tr>\n<tr style=\"height: 352px;\">\n<td style=\"width: 47.4197%; height: 352px;\"><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">It is a related technique, but instead of using antibodies to detect virus antigen, it uses virus antigen to detect antibodies. A positive ELISA indicates the presence of antibody to a virus in our patient.<\/span><\/p>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\"> That patient may have had a viral infection to which their <a href=\"https:\/\/www.hopkinsmedicine.org\/health\/conditions-and-diseases\/the-immune-system#:~:text=The%20immune%20system%20protects%20your,which%20you%20are%20born%20with.\" target=\"_blank\" rel=\"noopener\"><span style=\"color: #0000ff;\">immune system<\/span><\/a> has responded. Often, this will mean they have no live virus and will have recovered. Since antibodies can persist for a while after a virus infection has occurred, ELISA can detect infections that have occurred a while ago.<\/span><\/p>\n<p><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><b>\u00a0<\/b><\/span><\/td>\n<td style=\"width: 51.7373%; height: 352px;\"><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">It detects viral antigens (proteins usually on the <a href=\"https:\/\/praxilabs.com\/en\/blog\/2020\/02\/03\/biology-of-viruses-new-coronavirus\/\">surface of viruses<\/a>) using antibodies against those proteins. A positive Western blot indicates the presence of viral antigen \u2013 which very often means live virus \u2013 in our patient. That patient may have an ongoing viral infection.<\/span><\/p>\n<p><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><b>\u00a0<\/b><\/span><\/td>\n<\/tr>\n<tr style=\"height: 11px;\">\n<td style=\"width: 47.4197%; height: 11px;\"><\/td>\n<td style=\"width: 51.7373%; height: 11px;\"><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><b>\u00a0<\/b><\/span><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<h3><span class=\"ez-toc-section\" id=\"What_Does_a_Western_Blot_Tell_You\"><\/span><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><b>What Does a Western Blot Tell You?<\/b><\/span><span class=\"ez-toc-section-end\"><\/span><\/h3>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">A western blot is a laboratory method used to detect specific protein molecules from among a mixture of proteins. This mixture can include all of the proteins associated with a particular tissue or cell type. Western blots can also be used to evaluate the size of a protein of interest and to measure the amount of protein expression. This procedure was named for its similarity to the previously invented method known as the Southern blot.<\/span><\/p>\n<h3><span class=\"ez-toc-section\" id=\"How_Long_Does_Western_Blot_Test_Take\"><\/span><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><b>How Long Does Western Blot Test Take?<\/b><\/span><span class=\"ez-toc-section-end\"><\/span><\/h3>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Western blot tests take only one day to perform, but some laboratories may not run the test every day. Results from rapid tests done in the health care professional&#8217;s office or at other points of care are usually available in 15-20 minutes.<\/span><\/p>\n<h3><span class=\"ez-toc-section\" id=\"What_Is_the_Purpose_of_the_Transfer_in_Western_Blot_protocol\"><\/span><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><b>What Is the Purpose of the Transfer in Western Blot protocol?<\/b><\/span><span class=\"ez-toc-section-end\"><\/span><\/h3>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Western Transfer, also known as Western blotting, is a rapid immunoblotting technique for identifying the presence of a particular protein in a complex mixture of proteins such as cell lysates or sera. The technique exploits both the efficiency of SDS-PAGE to separate a mixture of proteins into distinct protein bands and the ability of immunochemical reagents to interact specifically with a given protein antigen.<\/span><\/p>\n<h3><span class=\"ez-toc-section\" id=\"Can_Western_Blot_Be_Quantitative\"><\/span><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><b>Can Western Blot Be Quantitative?<\/b><\/span><span class=\"ez-toc-section-end\"><\/span><\/h3>\n<p><span style=\"font-weight: 400; font-size: 14pt; font-family: tahoma, arial, helvetica, sans-serif;\">Western blot is a reliable quantitative method only if sample properties and integrity, antibody specificity to the target protein, and loading protocols are considered. With careful attention to details, you can avoid common mistakes and avoid misinterpreting Western blot data.<\/span><\/p>\n<h2><span class=\"ez-toc-section\" id=\"Western_Blot_PraxiLabs_Virtual_Lab\"><\/span><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\">Western Blot\u00a0 PraxiLabs Virtual Lab<\/span><span class=\"ez-toc-section-end\"><\/span><\/h2>\n<p style=\"text-align: center;\"><iframe loading=\"lazy\" src=\"\/\/www.youtube.com\/embed\/sldmSbygsSI\" width=\"560\" height=\"314\" allowfullscreen=\"allowfullscreen\"><\/iframe><\/p>\n<p style=\"text-align: center;\"><span style=\"font-family: tahoma, arial, helvetica, sans-serif; font-size: 14pt;\"><strong><span style=\"color: #0000ff;\"><a style=\"color: #0000ff; text-decoration: underline;\" href=\"https:\/\/praxilabs.com\/en\/sign-up\" target=\"_blank\" rel=\"noopener\">Create Your Free Account<\/a><\/span> and Try Our Simulation \u201cWestern Blot\u201d<\/strong><\/span><\/p>\n<p style=\"text-align: center;\"><span style=\"font-family: tahoma, arial, helvetica, sans-serif;\"><strong><a class=\"maxbutton-3 maxbutton\" href=\"https:\/\/praxilabs.com\/en\/sign-up\"><span class='mb-text'>Create a FREE Account Now!<\/span><\/a><\/strong><\/span><\/p>\n","protected":false},"excerpt":{"rendered":"<p>Western blot is an indispensable mechanism in the modern biomedical research laboratory, as well as in laboratories doing research in other areas. It is considered as an analytical technique used mainly in molecular biology and immunogenetics where antibodies are used to specifically detect their antigen. Before discussing how to analyze western blot data, you can &hellip;<\/p>\n","protected":false},"author":8,"featured_media":4449,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_lmt_disableupdate":"no","_lmt_disable":"no","footnotes":""},"categories":[6],"tags":[],"class_list":["post-907","post","type-post","status-publish","format-standard","has-post-thumbnail","","category-biology"],"modified_by":"Muhamed Elmesery","_links":{"self":[{"href":"https:\/\/praxilabs.com\/en\/blog\/wp-json\/wp\/v2\/posts\/907","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/praxilabs.com\/en\/blog\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/praxilabs.com\/en\/blog\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/praxilabs.com\/en\/blog\/wp-json\/wp\/v2\/users\/8"}],"replies":[{"embeddable":true,"href":"https:\/\/praxilabs.com\/en\/blog\/wp-json\/wp\/v2\/comments?post=907"}],"version-history":[{"count":23,"href":"https:\/\/praxilabs.com\/en\/blog\/wp-json\/wp\/v2\/posts\/907\/revisions"}],"predecessor-version":[{"id":4196,"href":"https:\/\/praxilabs.com\/en\/blog\/wp-json\/wp\/v2\/posts\/907\/revisions\/4196"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/praxilabs.com\/en\/blog\/wp-json\/wp\/v2\/media\/4449"}],"wp:attachment":[{"href":"https:\/\/praxilabs.com\/en\/blog\/wp-json\/wp\/v2\/media?parent=907"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/praxilabs.com\/en\/blog\/wp-json\/wp\/v2\/categories?post=907"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/praxilabs.com\/en\/blog\/wp-json\/wp\/v2\/tags?post=907"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}