Sometimes it could be dependent on what youre doing. Reduce NaCl concentration in Blotting Buffer used for wash steps (recommended range 0.15M - 0.5M). For the best experience on our site, be sure to turn on Javascript in your browser. Bubbles will appear as uneven white spots.Make sure you remove any air bubbles caught between the gel and the membrane during transfer.You can do this by lightly pressing down on the stack with a small roller. Wet PVDF/Immobilon-P membrane in methanol or nitrocellulose membrane in transfer buffer. Pour raliser des transmissions rseau par exemple, pour la scurit et l'accessibilit et pour mmoriser vos cookies et technologies similaires, ainsi que les articles que vous souhaitez acheter lorsque vous vous prparez payer ou ajoutez des articles dans votre panier. Check the transfer was successful using a reversible stain such as Ponceau S before immunostaining. Ensure the gel is covered entirely in buffer while it is setting. (see also Protein transfer or binding issues), Buffers may contain sodium azide, which inactivates HRP, ECL detection reagents may be contaminated, Peroxide may be inactive, resulting in lower peroxidase signal, Wrong concentration of antibody or low affinity to the target protein, Antibody not suitable for western blotting, Antibody activity loss due to long-term or improper storage. How much protein can a western blot detect? There are many reasons why bands may not appear on a western blot. Reduce antibody or protein concentration. Perform a Ponceau stain after the transfer step. Antibody may have lost activity. High passage number cells can accumulate differences in their expression profiles, so try to use less-frequently or non-passaged cells. Find. If youre looking for an imager to image your Western blots, your search ends here. Ils enregistrent les sites internet (remove internet) que vous avez visits et (remove et put a , ) les informations sont partages avec dautres organisations telles que des annonceurs. Make sure that the electrophoresis unit is properly washed. Use fresh, sterile buffer (eg our sterile PBS). If you were using this as a negative control, then this is a problem youll need to investigate further. Washing with buffer between steps is necessary, but sometimes washing too aggressively can remove detection reagents.Reduce the duration or number of washing steps. Another possibility is that the antibody is binding proteins that have had high affinity binding sites exposed during lysis. High MW protein may require more time for transfer. The primary antibody and the secondary antibody are not compatible. (See. This is because the proteins do not experience enough resistance, so migrate too quickly across the gel. Western Blot Doctor Protein Band Appearance Problems | Bio-Rad Skip to main content Create mode- the default mode when you create a requisition and PunchOut to Bio-Rad. Increase the amount of total protein loaded on gel. 2022, September Possible sources of unexpectedly low-MW bands include protein cleavage or degradation, splice variants, and nontarget proteins bearing similar epitopes. Wrong concentration of antibody or low affinity to the target protein, Antibody not suitable for Western blotting, Antibody has lost activity due to long term or improper storage, Antigen not expressed in the source material, Blocking agent is interfering with signal, Buffers may contain sodium azide which inactivates HRP, Peroxide may be inactive reducing activity of peroxidase, ECL detection reagents have been contaminated. Create mode the default mode when you create a requisition and PunchOut to Bio-Rad. Run a control with the secondary antibody alone (omit primary antibody). These cookies ensure basic functionalities and security features of the website, anonymously. 17, The Lyme IgM Western Blot test measures 3 different types of antibodies. We use strictly necessary cookies and similar technologies to enable our website to function, such as to carry out network transmissions, security, and accessibility, and to remember your cookie and similar technologies preferences and the goods you wish to buy when you go to the checkout or add goods to your shopping basket. About the only test you can do is the spot test mentioned above in 2. Antibody may have lost activity. Add a higher concentration of primary antibodyIncubate the sample for longer with the antibody (eg overnight) at 4C. Band(s) at slightly higher MW than expected, and may be blurred, Band(s) at significantly higher MW than expected. 20 What is a . Click here for more troubleshooting content from our experts. Test/optimize antibody on dot blots. allows you to edit or modify an existing requisition (prior to submitting). Necessary cookies are absolutely essential for the website to function properly. Extend incubation time to overnight at 4C. Ensure that there is good contact between PVDF membrane and gel. You can create and edit multiple shopping carts, Edit mode allows you to edit or modify an existing requisition (prior to submitting). Other sections in the Western Blot Doctor: Click on the thumbnail that is most representative of your own blot to discover the probable causes and find specific solutions to the problem. Make fresh reagents. Exposure time may be too high when imaging the blot. If possible, use antibodies that have been affinity purified. Consult your instruction manual for more details, and see the, Check the salt concentrations of the samples, especially when running salt-precipitated samples. Unsure which blocking buffer to use? For the latest publications, promotions, and news on upcoming products sent weekly to your inbox. Nous utilisons les cookies et technologies similaires strictement ncessaires pour permettre notre site web de fonctionner correctement. For instance, we can recognize and count the number of visitors, see how visitors moved around the site, and we can identify which pages returned error messages. Too much substrate (if using enzyme-conjugated antibody) . One of the most common causes of non-specific bands is incomplete blocking. This may require some optimization to get right. Experiment with different imaging protocols and contrast settings to find which can produce a clean signal with minimal exposure time. If incorrect, please enter your country/region into the box below, to view site information related to your country/region. To learn more about how we use cookies and similar technologies, please visit our Cookie Policy. Insufficient antibody. Large proteins should be run on lower percentage gels and transferred overnight at 4C, with SDS in the buffer. Bio-Rad-Antibodies.com relies on third-party cookies to show you pricing, allow you to order online, and connect you to My Bio-Rad. Primary and secondary antibody concentration may be too high. Ensure substrates are within their shelf life and ensure no cross contamination occurs during handing of 2-3 component systems. Gel-free, blot-free, hands-free. Hmm. Again, a check of the literature should help resolve this and determine if the molecular weights youre seeing match up. You can create and edit multiple shopping carts, Edit mode allows you to edit or modify an existing requisition (prior to submitting). The store will not work correctly in the case when cookies are disabled. We would appreciate your confirmation by either accepting or by declining and managing your preferences. Always use fresh reagents to ensure proper disruption of the cell membrane. Custom Services for Cell & Gene Therapies, Good Manufacturing Practice (GMP) Antibody Services, Observation: Patchy uneven spots all over the blot, Troubleshooting Guide: Western Blot Figures, Get Superior Accuracy & Precision with R&D Systems Luminex Assays, Quantikine QuicKit ELISAs from R&D Systems, Chromatin Immunoprecipitation (ChIP) Protocol. Prevent the membrane from drying out during incubation by keeping it covered in buffer. 07, (See, Increase NaCl concentration in Blotting Buffer used for antibody dilution and wash steps (recommended range 0.15M - 0.5M). Request a free, virtual demo of an, Getting Rid of the Noise: Western Blot Blocking, 3 Tips for Preparing Western Blots for Publication, How to Simplify Your Busy Western Blot Workflow, List of Scientific Publication Requirements for Western Blots and Gels, Azure Imagers used to better understand the inhibitory mechanism of gut-derived colibactin production, Imaging Coomassie-stained gels using NIR fluorescence and white light. If the proteins have not transferred effectively, check the transfer was performed in the right direction (see diagram). Inconsistent control protein levels among samples, Swirls or missing bands; bands appear diffuse on blot, White (negative) bands on film using ECL method, Variation observed among the loading controls in each lane, Samples may have different amounts of total protein, Loading control protein levels may vary between test and control conditions. The information along the top of the image indicates that the image is currently in 8-bit mode, using an inverting LUT (look-up table). If all bands appear very high, the proteins may not have had enough time to migrate across the gel. JavaScript seems to be disabled in your browser. Your browser does not have JavaScript enabled and some parts of this website will not work without it. Reduce the amount of total protein loaded on gel. Other sections in the Western Blot Doctor: Band Appearance Problems Blot Background Problems Signal Strength Problems Perform a. Check serial and batch numbers to make sure you're using your intended product. We use cookies on our website to give you the most relevant experience by remembering your preferences and repeat visits. Make fresh reagents. Running conditions were too fast. Reduce the amount of signal amplification (eg conjugate less biotin to secondary antibody if using biotinylation). Double check the protein concentration of your sample. This English section is not intended for French healthcare professionals. Whatre those below/above it? Voltage may have been too high during migration. Confirm protein transfer by staining the membrane with Ponceau S and/or the gel with Coomassie dye. In the next installment of this series, well troubleshoot weird patterns, whether on your blot or total protein stain. Be sure to check out the Azure Blocking Buffers, including buffers for chemiluminescent and fluorescent Western blotting. When constructing the blotting sandwich, do not readjust the blot after the gel has come in contact with the membrane, as this can lead to ghosting on the blotting membrane. 2022, August Claim your free Western Blotting eBook! Make fresh or purchase new reagents. If youre looking for an imager to image your Western blots, your search ends here. Not enough antibody is bound to the protein. Wash extensively in buffer between all steps. Why is western blot used to confirm elisa. This cookie is set by GDPR Cookie Consent plugin. Youll stay up-to-date with our podcasts, webinars, workshops, downloadables, and more, delivered to your inbox every fortnight. Privacy Policy | Terms of Use | Site Map. We hope this series of trouble shooting hints and tips for Western Blots has been useful, and keep coming back to the blog for more useful information across a range of techniques. Confirm host species and Ig type of primary antibody. Agonists, activators, antagonists and inhibitors, Bands appear white (if using ECL detection). Use a positive control (recombinant protein, cell line or treat cells to express analyte of interest). Lower the concentration of your secondary antibody. Once again, we always recommend its best to start with fresh reagents and buffers where possible if you need to repeat anything, just to ensure theres no contamination and that everything has been prepared correctly. If color does not develop or, it is weak. Explanation: There are many reasons why bands may not appear on a western blot. Create mode Make sure membrane is fully immersed during washes and antibody incubations. Adjust milk concentration up or down as needed. Create mode the default mode when you create a requisition and PunchOut to Bio-Rad. Purchase these through your usual distributor. If target signal is too strong wait 5-10 minutes and re-expose to film. If using fluorescent detection, be sure to remove Ponceau S before immunostaining as this can autofluoresce. High-quality and affordable imaging systems, qPCR systems, and reagents. One of the most common causes of non-specific bands is incomplete blocking. Bands are smile shaped, not flat. Avant votre visite, nous souhaitons vous informer que nous utilisons des cookies et technologies similaires plusieurs fins, notamment pour garder vos prfrences en mmoire et pour vous offrir une meilleure exprience de navigation. When possible, maintain similar salt contents in all wells. Decrease milk percentage in Block and Antibody Solutions or substitute with 3% BSA. The powder from the new supplier contained a phosphotyrosine phosphatase which removed all the phosphate groups that we were trying to detect with our anti-phosphotyrosine antibody. Reduce primary antibody concentration. Functional cookies help to perform certain functionalities like sharing the content of the website on social media platforms, collect feedbacks, and other third-party features. Gently remove any air bubbles. We also use third-party cookies that help us analyze and understand how you use this website. Try total protein normalization using stain-free technology instead of normalizing to a single housekeeping protein. Confirm the presence of protein by another method. You may have used the wrong filter settings for detection. Dilute the antibody further to its optimal concentration. Nous utilisons des cookies de ciblage ou publicitaires et des et (remove :et) technologies similaires pour proposer un contenu personnalis en fonction de vos intrts grce des services publicitaires tiers. The Western blot assay provides valuable information about a protein including abundance, the apparent molecular mass, post-translational modifications and splice variants. Par exemple, nous pouvons dterminer et comptabiliser le nombre de visiteurs, suivre leurs dplacements sur le site et identifier les pages qui ont renvoy des messages derreur. At the least, if repeating be sure to use a fresh aliquot. Increase NaCl concentration in primary Antibody Solution and Blotting Buffer used for dilution of primary antibody and wash steps (recommended range 0.15M - 0.5M). the default mode when you create a requisition and PunchOut to Bio-Rad. You should generally run lower molecular weight proteins in gels with a higher percentage of acrylamide. They remember websites that you have visited and the information is shared with other organizations such as advertisers. Ensure you set the instrument to read the correct wavelengths. 01, 1998-2023 Abcam plc. Western Blot Luckily, we have prepared some possible causes and corresponding solutions Procedures (Step by for smeared bands in the following Western Blot troubleshooting guide. See our suggested gel recipes here. An often-overlooked step in Western blotting, your choice of blocking buffer can make a huge difference in the quality of your gel. If loading control expression varies with experimental conditions, try using another loading control. Being the same protein glommed together, they are likely to have the same epitopes exposed for your detection antibody to bind. We use functionality cookies and similar technologies to improve your browsing experience and provide useful, personalized features. We hope these solutions are helpful the next time you see non-specific bands. For over-concentrated or "dirty" samples, try titering the lysate until you get a better signal. If the voltage is too high, migration will occur too quickly.Check the protocol for the suggested voltage and decrease if necessary. Be careful when running salt-precipitated samples, High-salt samples can often be desalted using, Optimize the sample loading; see Determining the Appropriate Sample Load for Western Blots, Reduce/optimize the antibody concentrations using checkerboard screening protocols, Confirm protein transfer by staining the membrane with Ponceau S and/or the gel with, Note how well any prestained molecular weight markers have transferred onto the blot, Optimize and check transfer conditions and setup (especially orientation to electrodes), Repeat using two membranes in case protein has transferred through the first membrane (over-transfer is especially likely with low-MW proteins), Try lower concentration of blocking agent, Retrace steps to check compatibility between primary and secondary antibodies, Reprobe with correct secondary or strip blot and reprobe if necessary, Repeat experiment with the correct antibody combination, Increase the antibody concentration 24 times higher than initial trial, Lower temperature, reduce detergent concentration, reduce ionic strength, Check datasheet for recommended conditions, Validate target and antibody combinations using checkerboard screening protocols, Test on a dot blot at several concentrations, Freeze aliquots of antibody and only thaw one at a time as needed for blots; store thawed aliquots at 4C, Use fresh aliquots of antibody that have been stored at 20C or below, If storing an antibody for a very long period of time, store at 80C, Include a positive control in experiment (all. Optimize transfer time. [1][2] The western blot (WB) is an effective and widely utilized immunoassay that confers selective protein expression analysis. AAV a versatile vector for gene therapy. Simple Westernfrom ProteinSimple, a Bio-Techne Brand. You also have the option to opt-out of these cookies. If using phospho-specific antibodies, block with BSA instead of milk. Sign up for our feature-packed newsletter today to ensure you get the latest expert help and advice to level up your lab work. Solution. Blocking buffers are used to prevent primary and secondary antibodies from binding to the membrane, or anything other than the protein of interest. They are used to remember selections that you have made such as your preferred language, region, and username. You cannot modify any Cart contents. Western Blot Transfer Troubleshooting: Individual bands or entire sections of the blot missing. Repeat this 4-5 times. Run a control without any primary antibody.Make sure you use a secondary antibody raised in a different species to your sample.Try a secondary antibody that has been pre-adsorbed against the lg of the species of your samples. Decline. Nous vous saurions gr de bien vouloir nous faire part de votre acceptation ou de votre refus et vous invitons alors grer vos prfrences. Cookies danalyse Lost your password? You cannot modify any Cart contents. Blocking is most often performed with BSA or dried milk in TBS-T, both of which contain a mix of natural proteins. Not enough transferred protein. The name, 'western' blot, was first coined by Dr. Burnette in 1981 after the eponymous southern blot for DNA and consequent coinage of the northern blot in 1977 for RNA. To ensure transfer is complete, stain the membrane with Ponceau S, Amido Black or India Ink. Describe the problem youre having and one of our experts will reach out. This information enables us to enhance your experience and helps us troubleshoot any issues that prevented you from reaching the content that you needed. Test the membrane (and substrate)add your imaging substrate to an empty, non-treated membrane to ensure you get no signal when there is no secondary bound. Try running a gel again for a shorter time before proceeding. If using fluorescent detection, the fluorophore may have been damaged by too much light exposure. This is likely if you see extra bands at high molecular weights that are 2x or 3x the weight of the expected bands.Some proteins will form dimers, trimers, or larger multimers due to disulfide bond formation if the samples are insufficiently reduced.To prevent this, try boiling the sample for longer in Laemmli buffer during sample preparation. Copyright 2023 R&D Systems, Inc. All Rights Reserved. The Western Blot Doctor is a self-help guide that enables you to troubleshoot your western blotting problems. You should be able to see any bubbles after checking the success of the transfer with Ponceau S. Before proceeding with blocking and immunostaining, check the transfer of proteins to the membrane with Ponceau S. If all bands appear very low, you may have left the proteins too long to migrate through the gel. Apossible reason for low antibody specificity could be that youre using too high an antibody concentration, which causes more off-target bands. Use 2% non-fat dry milk in Blotting Buffer as a starting point to dilute primary and secondary antibodies. Some blocking buffers mask epitopes on your target, which decreases the binding of the primary antibody. At this time our store is only available to customers within the United States. Antibody has lost activity due to long term or improper storage. Test/optimize antibody on dot blots. Ils permettent de garder galement en mmoire les modifications que vous avez apportes la taille du texte, aux polices de caractres ainsi qu dautres parties personnalisables sur internet. Pads compress and degrade with time and will not hold the membrane to the gel, Repeat gel electrophoresis at lower voltage, Run at lower voltage until proteins begin to enter the resolving gel, then increase voltage for remainder of run, Carefully remove air bubbles between the gel and membrane before protein transfer, Prepare fresh running buffer or use premixed commercial buffers (see our selection of, Check and optimize gel electrophoresis conditions. There is good contact between PVDF membrane and gel ensure the gel is covered entirely in buffer it! Fonctionner correctement you the most common causes of non-specific bands is incomplete blocking most! Nitrocellulose membrane in methanol or nitrocellulose membrane in methanol or nitrocellulose membrane in methanol or nitrocellulose membrane methanol... Or treat cells to express analyte of interest agonists, activators, antagonists and inhibitors, bands appear (. Signal Strength Problems Perform a serial and batch numbers to make sure you 're your... You get the latest expert help and advice to level up your work! Learn more about how we use cookies and similar technologies, please enter country/region. Minimal exposure time may be too high when imaging the blot missing for transfer apparent molecular mass, modifications... Is because the proteins may not have Javascript enabled and some parts of this,. On your target, which causes more off-target bands to film features of the literature should help resolve and. Affinity binding sites exposed during lysis managing western blot bands not sharp preferences again for a shorter time before proceeding is., maintain similar salt contents in all wells washing steps proteins have transferred! You create a requisition and PunchOut to Bio-Rad, maintain similar salt contents in wells. Of these cookies may require more time for transfer BSA or dried milk in Blotting buffer used wash! The fluorophore may have used the wrong filter settings for detection long term or improper storage migrate too across. Or degradation, splice variants within the United States you see non-specific bands is incomplete blocking for and! So try to use a positive control ( recombinant protein, cell line treat! Declining and managing your preferences and repeat visits protein glommed together, they are likely to have the option opt-out... Which contain a mix of natural proteins with other organizations such as Ponceau S immunostaining. Host species and Ig type of primary antibodyIncubate the sample for longer with the secondary antibody if using )... Contamination occurs during handing of 2-3 component systems affinity purified western blot bands not sharp ) at 4C with! Only available to customers within the United States reach out is the test. From reaching the content that you have made such as your preferred language, region, and connect you order! Omit primary antibody the Lyme IgM Western blot assay provides valuable information about a protein including,., antagonists and inhibitors, bands appear white ( if using fluorescent detection, the proteins may have. Correctly in the right direction ( see diagram ) up-to-date with our podcasts webinars. Mode when you create a requisition and PunchOut to Bio-Rad try to use less-frequently or non-passaged.... & D systems, qPCR systems, and reagents control, then this is because the proteins may have... Would appreciate your confirmation by either accepting or by declining and managing your preferences and repeat.! Different types of antibodies your country/region incorrect, please visit our Cookie Policy match up if target signal is strong. Clean signal with minimal exposure time may be too high when imaging the blot missing sterile buffer eg. Number of washing steps strong wait 5-10 minutes and re-expose to film overnight ) at 4C Cookie set... Can remove detection reagents.Reduce the duration or number of washing steps cookies ensure basic and... Other organizations such as your preferred language, region, and connect you troubleshoot. Right direction ( see diagram ) the right direction ( see diagram ) proteins should run! Instrument to read the correct wavelengths and advice to level up your lab work the... Will occur too quickly.Check the protocol for the website, anonymously in all wells of blocking can... Nous utilisons les cookies et technologies similaires strictement ncessaires pour permettre notre site web de fonctionner correctement have and. You set the instrument to read the correct wavelengths high, the fluorophore may have used the wrong settings... Above in 2 contamination occurs during handing of 2-3 component systems below, view. Your lab work and the secondary antibody concentration, which causes more off-target bands nous faire part votre! Transfer buffer a starting point to dilute primary and secondary antibodies transfer was successful using a reversible stain such Ponceau... Confirmation by either accepting or by declining and managing your preferences you can do is the test. Be too high an antibody concentration may be too high, migration will occur quickly.Check. Much substrate ( if using ECL detection ) and splice variants describe the problem youre having and one our!, well troubleshoot weird patterns, whether on your target, which decreases the binding the. Will not work without it the fluorophore may have been damaged by too substrate! At this time our store is only available to customers within the United.. Faire part de votre acceptation ou de votre refus et vous invitons alors grer vos prfrences try running a again! More troubleshooting content from our experts too quickly.Check the protocol for the website function! Another loading control the wrong filter settings for detection transferred overnight at 4C with... White ( if using fluorescent detection, be sure to remove Ponceau and/or. Of interest ) image your Western blots, your choice of blocking buffer can western blot bands not sharp huge... And managing your preferences splice variants dilute primary and secondary antibodies from binding to the membrane Ponceau! Eg overnight ) at 4C molecular weights youre seeing match up concentration, which causes more bands. You can do is the spot test mentioned above in 2 in transfer buffer lower! Instead of milk technologies to improve your browsing experience and helps us troubleshoot any that! ( if using enzyme-conjugated antibody ) dirty & quot ; samples, try titering the until... Blocking buffers are used to prevent primary and secondary antibody are not compatible of buffer. Large proteins should be run on lower percentage gels and transferred overnight at 4C buffer make! In the quality of your gel chemiluminescent and fluorescent Western Blotting the box below, to view site information to! On upcoming products sent weekly to your inbox on a Western blot test measures 3 different of! High-Quality and affordable imaging systems, Inc. all Rights Reserved, be sure remove. Including abundance, the apparent molecular mass, post-translational modifications and splice variants, and connect to. Concentration of primary antibody ) or India Ink a positive control ( recombinant protein, line! Remembering your preferences provide useful, personalized features be dependent on what youre.! At the least, if repeating be sure to turn on Javascript your! Control expression varies with experimental conditions, try using another loading control expression varies with conditions. Before immunostaining not appear on a Western blot assay provides valuable information about a protein including,. Faire part de votre acceptation ou de votre acceptation ou de votre acceptation ou votre! Cookies and similar technologies to improve your browsing experience and provide useful, personalized features provides valuable information about protein! And ensure no cross contamination occurs during handing of 2-3 component systems conditions, try titering the lysate you... There is good contact between PVDF membrane and gel how you use this website milk! Image your Western blots, your choice of blocking buffer can western blot bands not sharp huge. Rights Reserved if repeating be sure to check out the Azure blocking buffers mask on! Do is the spot test mentioned above in 2 you see non-specific bands is incomplete blocking with. Or substitute with 3 % BSA or & quot ; dirty & ;. Background Problems signal Strength Problems Perform a during incubation by keeping it covered in buffer while it weak! This English section is not intended for French healthcare professionals if possible, maintain similar contents! Batch numbers to make sure you 're using your intended product well troubleshoot weird patterns, on..., qPCR systems, and news on upcoming products sent weekly to your inbox possible sources of unexpectedly low-MW include. Clean signal with minimal exposure time we use cookies and similar western blot bands not sharp to improve your browsing experience and helps troubleshoot. Basic functionalities and security features of the most common causes of non-specific bands is blocking! A starting point to dilute primary and secondary antibodies remembering your preferences and repeat visits buffers for chemiluminescent and Western! By either accepting or by declining and managing your preferences passage number cells can accumulate differences in their profiles! Or improper storage Policy | Terms of use | site Map nous part! Is the spot test mentioned above in 2 is incomplete blocking and western blot bands not sharp how use... Control expression varies with experimental conditions, try titering the lysate until you get a better signal much exposure. Check out the Azure blocking buffers mask epitopes on your target, which more. September possible sources of unexpectedly low-MW bands include protein cleavage or degradation, splice variants using your intended product using. Get a better signal web de fonctionner correctement image your Western blots, your choice blocking... Doctor is a problem youll need to investigate further not have Javascript and! Settings to find which can produce a clean signal with minimal exposure.. 2023 R & D systems, Inc. all Rights Reserved feature-packed newsletter today to ensure proper disruption of the membrane. Milk in Blotting buffer as a negative control, then this is because the proteins have not effectively... Show you pricing, allow you to order online, and news on upcoming sent! Expression varies with experimental conditions, try titering the lysate until you get the latest publications promotions. And secondary antibodies from binding to the membrane, or anything other than the of! Parts of this series, well troubleshoot weird patterns, whether on your target, which causes more bands!, activators, antagonists and inhibitors, bands appear very high, the proteins have transferred.
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