Western Blot

Western Blot

  • Terra Elizabeth Wolfe added an answer:
    What is the best way to strip a Western Blot?

    Does stripping with only glycine, SDS and tween tend to leave a background signal?

    Terra Elizabeth Wolfe · North Carolina Central University

    The commercial stripping buffer I had from ThermoSci was too weak for my needs. I made my own. It's more harsh but it did the job. Protocol attached. Recipe:

    Stripping Buffer:
    For 100 mL
    • 20 ml SDS 10%
    • 12.5 ml 0.5 M Tris HCl pH 6.8
    • 67.5 ml ultra-pure water
    Add 0.8 ml ß-mercaptoethanol under the fume hood

  • Ketil winther Pedersen added an answer:
    Can anyone explain why I am not seeing heavy and light chains but seeing the protein of interest in IP?

    I am doing an IP and got a peculiar result. I pulled protein X, ran it on a gel, and probed for protein X. I used the same antibody for the IP and the western. I got my protein of interest at the correct molecular weight, but the expected antibody heavy and light chains were conspicuously absent. I used a different antibody for the same experiment and got protein X and the heavy and light chains. Why am I not seeing these heavy and light chains in my IP when I use that first particular antibody?

    Ketil winther Pedersen · ThermoFischer Scientific,


    in terms of heavy and light chain on your blot this can be "removed" by using the TrueBlot secondary Ab (HRP) instead of regular secondary Abs. these will not recognize heavy or light chain on the membrane while labeling. we have used it for a long time and they work very well.
    If the primary Ab is covalently coupled to beads such as Dynabeads less will be eluted off if mild elution is used (e.g. low pH). This in combination with TrueBlot have worked very well in our hands as we are working with targets at similar size as heavy or light chain.

  • Matthew William Turnbull added an answer:
    Why does my flag-tagged exogenous protein give multiple bands?
    In my western blot analysis of both total cell lysate and immunoprecipitated samples, I saw 2 bands. The expected band is around 15kDa but I see another additional band around 25kDa in every western blot that I performed. The protein that I am working on is flag tagged. I thought it might be a post-translational modification, but since the difference is 10kDa, it is unlikely. Additionally, the protein is an endosomal protein. If you have any idea about the reason please help me.
    Thank you.
    Matthew William Turnbull · Clemson University

    Have you looked at the possibility that you have dimers present? Stronger denaturation or reducing conditions might help avoid this possibility.

  • Katrin Schweitzer added an answer:
    How do I remove IgG band covering my protein sample while doing a western blot for ChIP?

    I am optimising ChIP for my protein which is approx. 45kDa in size. Upon doing Western Blot to check my pulldown I see a big IgG band that covers up my protein. Can anyone suggest me how to resolve this? qPCR is another option but I want to first see the pulldown by Western blotting.

    Also, can some one suggest me secondary antibodies that do not recognise the IgG have chain that I can use to avoid this situation?

  • Salman Malakpour added an answer:
    How do you do your western blot normalization ?


    I'm working on a new project which involve "quantify" an effect on protein levels. As I am working with rat samples I have a lot of samples to pass through Western blot.

    In my previous lab I never had the chance to perform this step so right now I have a lot of questions. I wonder if I can compare different blots and how ?

    And I have to say that for some of my proteins I use different exposition time for the same membrane (I cut the membrane in different pieces) and depending on saturation. I try to be at the maximum signal before any saturation but for some of my protein of interest I can say that it will never be the case (signal too faint). Am I doing right ?

    Should I present my result as ratio of protein of interest on loading control and only then compare them. Or should I compare membrane per membrane and then mix all the results ?

    I don't know if I am being clear...

    Thank you

    Salman Malakpour · University of Porto

    Hi  Dieynaba Ndiaye
    Just in case I would suggest to prepare the pool for each set of your tissue sample, before rinning your experiment samples, for example Kidney, liver, brain poll etc as follow: add same equal amount of each sample (for example 25ul or more) to one eppendorf tube and making pool. You can use prepared pool first to test different antibody then use your original samples to save more of your samples.

    Regarding the normalization, the B actine might be used for comparation.

    Good luck!


  • Ketil winther Pedersen added an answer:
    Did I immunoprecipitate my protein?

    Hello colleagues,

    This is one of my first IPs so I am a bit confused with the result I just obtained.

    I used the dynabeads protein G for my purposes.

    Here it is, my protein is ~97kDa (in the red box) and it seems that I have it in the IP, however when I screen that IP's supernatant, negative control supernatant (that I IP'd with irrelevant IgG) and just the cell lysate I see almost at the same level (but a little higher) the protein that I am actually trying to IP ...

    So here I am not sure that what's been IP'd is actually something that may interest me. (it is interesting but maybe it's just not an IP signal)

    Shouldn't the IP and control signals be at the same level?

    Or it is a normal situation? If yes, why that happens?

    Thank you!

    Ketil winther Pedersen · ThermoFischer Scientific,


    during the elution of your target, some capture Ab on your Dynabeads will also be eluted off. They will show up on your blot after staining the blot. One way of removing these bands is to use the TrueBlot secondary Ab (HRP coated) which will not recognize the heavy or light chain at all. I have used it many times as we are working with molecules with the size around 50 kDa or 25 kDa.

    kind regards


  • Priyanka Firmal added an answer:
    When running pentavalent IgM through SDS-PAGE, how many bands would be obtained by western blotting?

    If a pentavalent IgM is run through SDS-PAGE, how many bands would be obtained by western blotting using alkaline phosphatase conjugated secondary antibody?

    Priyanka Firmal · Institute of Microbial Technology

    Thank you sir for the prompt replies.Yes@ Indramohan Sir,this question is from CSIR-NET exam and i found that the information given is incomplete.I also got confused as against which region they are targeting the secondary antibody.I marked the option which says 3 bands should be observed as there would be three bands on SDS-PAGE (H,L,J chains) and secondary ab could be targeted against any of the three or in a broader perspective all the three.

  • Atif Baig added an answer:
    Can someone suggest any antibodies against mouse TNRC6A, TNRC6B and TNRC6C?

    Hi everyone

    I appreciate it if you would inform me which antibodies are good to detect mouse GW182 proteins, TNRC6A, TNRC6B and TNRC6C by western blotting.

    Are they commercially available?

    Thank you very much in advance!

    Atif Baig · Universiti Sultan Zainal Abidin | UniSZA


  • Angel Flores added an answer:
    Does anyone ever reuse Thermo's Superblock multiple times for your western blots?

    Our lab is using the Superblock reagent from Thermo Scientific. It's an expensive reagent but we like to use it because it does an excellent job at blocking. I block for 15 minutes at room temperature and move on to the next step. I have been discarding the Superblock after using it each time. However, I am curious if this reagent can be used more than once.

    Angel Flores · University of California, Los Angeles

    Thanks. I appreciate the input.

  • Heidi Kaastrup Müller added an answer:
    Does anyone have experience using soy milk as a blocking agent for IR western blotting?

    We recently started using a Licor Odyssey Fc machine and got great results with the blocking buffer that Licor provides with the machine. However, we have used almost all of it and it is really expensive...so we tried our old recipes we used for our kodak imager/chemi that is simply non fat dry milk, tween, diH20 and TBS. Unlike previous results with the Licor BB (pure black background with distinct bands) my bankground was extremely red. I started researching other alternatives such as fish gelatin etc and came across soy milk...anyone have any experience with this???

    Heidi Kaastrup Müller · Aarhus University Hospital

    I have attached the image showing four different blocking conditions: 5% Milk in TBS, 5% BSA in TBS, Odyssey Blocking Buffer/TBS (1:1) and Odyssey Blocking Buffer /TBS (1:2) on nitrocellulose membranes probed with mouse anti-actin and goat anti-mouse800CW. They were all really nice with no background, however, the actin bands were weaker in Milk and when tested with the red channel (700nm), Milk gave rise to autofluorescence.

  • Rania Ali added an answer:
    Is anyone familiar with Type I collagen detection in Western blots?
    Can anyone tell me one good buffer which works best to isolate type I collagen from murine skin samples to detect it through western blots?
    I have homogenized skin tissue and lysed in NP40 (normal lysis buffer) and also boiled directly the skin homegenate in BSB, in both the cases i could not see Type I collagen bands. Please suggest.
    Rania Ali · Case Western Reserve University School of Medicine

    Try to prepare your samples under native non reducing conditions

  • Shivam Mishra added an answer:
    I have been trying to use HeLa cell and lipofectamine 2000 to do a transfection, but somehow the band on the western blot is very light.
    I trited to optimize the system by using 2500ng of plasmid DNA with different amounts of lipofectamine 2000 (5ul, 7ul, 8ul, 9ul, 10ul) in the 6-well plate. There is a light band showed up in the well when I used 8ul and 9ul of lipofectamine. However, the intensity was as low as the background. Can anyone help me to optimize the transfection system? By the way, the protein size I tried to transfer is 65 K.
    Shivam Mishra · Agency for Science, Technology and Research (A*STAR)

    Hi Neil,

    Thanks for sharing about the PEI protocol. I will be very grateful to you if you can share it with me , at blue.shankar@outlook.com

    My Best,


  • Anand Shah added an answer:
    Can anyone help with a problem with Drp1 S637 antibody for western blotting?
    I have a problem using Drp1 S637 antibody from cell signaling for western blotting technique. Although it should be at around 70-80kD, I get a band at 120KD. Is this a splice version or a dimer?
    Anand Shah · Imperial College London

    Hi Andre,

    I've just used the same antibody looking for S637 p-DRP1 from human MDMs and have got the same size bands as well. The total protein looks fine at around 80 but the phospho-DRP1 gives a band over 100. I'm planning to run some extracts with a cAMP agonist I isolated previously as a positive control but was hoping you had managed to come up with an answer as to whether this is a non-specific band or the genuine protein in a homo-oligomer etc..

    Would be grateful for your help!

  • Penelope M Tsimbouri added an answer:
    Why does the western blot not show Input band in the IP-blot while the IP is positive?

    Hello guys,

    This might seem to be a silly question but for the past long time I have been phasing this issue in which control or the Input band just vanishes in the blot while I can clearly see the immunoprecipitated sample. If I run the the gel without the IP sample it works alright. The antibody in question is working properly. Since, the proteins are tagged. I have used antibodies both against the tag and the protein of interest, with the same result. I have tried different concentrations of protein in the input ranging from 20 ug to 100ug. Any suggestions or critique are welcome, thank you

    Penelope M Tsimbouri · University of Glasgow

    hi, i do agree with the saturation idea.

    one of my students had a similar problem and we ended up doing a titration of the sample, both the control and IP.

    in our case we needed 500micrograms of protein for the IP to get a signal from the transgene construct in the transgenic samples in comparison to the positive control which we only needed 10 micrograms. The band was always stronger in the positive controls and could not detect it in the samples. if we reduced the amount of control relative to the IP then the signal was much weaker as all the reaction was so much stronger with the IP sample.

    So the solution would be titrating your reactions to find the optimal amounts to load.

    good luck

  • Saemee Song added an answer:
    What are these streaks on silver stained polyacrylamide gels?

    Any good ideas as to what is causing these streak would be helpful. Thanks


    Second step of a taptag purification

    Bis-Tris Precast Polyacrylamide gels

    Ran at 165 volts

    1ul sample/20ul H2O/7ul 4x loading dye/ ~37mM DTT

    heat and spin samples before loading between 4-10ul to gel

    Saemee Song · Seoul National University

    I think, It might be due to a high voltage during electrophoresis. It is helpful to decrease the voltage and use a fresh running buffer. 

  • Jonathan T Ting added an answer:
    Does anybody know of a good antibody for tdTomato?

    I have a transgenic mouse with a tdTomato-tagged protein, so now I would like to immunoprecipitate the tdTomato and everything that is in the complex. Three antibodies (anti-DSRed) I tested result in an awful lot of background on western blots and also on tissue sections. Does anyone know of a good antibody? Thanks, Tineke

    Jonathan T Ting · Allen Institute for Brain Science

    Julien is right...the Rockland anti-RFP antibody is the best one we have tried for detecting tdTomato.  We made several transgenic mouse lines with tdTomato and used this antibody for all our characterization work.  The main issue is the photobleaching of native tdTomato, so it is much better to amplify the signal with the anti-RFP primary and use a more stable fluorecence-conjugated secondary antibody.  I often liked to use alexa-488 conjugated secondary to change the native red fluorescence to a green signal.  This gave me better resolution of fine structures like axonal projections.

  • Simone Tamburri added an answer:
    Can I store blotting paper after treating with ECL pierce, in case I want to develop it later on with X ray film?

    How long can I wait to develop an x ray film after my blotting paper is treated with ECL pierce (Chemiluminescence).

    Simone Tamburri · Ospedale di San Raffaele Istituto di Ricovero e Cura a Carattere Scientifico


    I suggest to you to incubate agacondary Ab for 30 min and ahe ECL.  I didn't see any signal after 1h of ECL incubation. So I think you have to expose the nitro to x ray within 30 min or again you can store the nitro in TBST added with NaN3 and then incubate again the secondary ab whenever you want

  • Jonathan Mark Wilson added an answer:
    Does anyone use anti-foam A to avoid foaming of samplings during homogenization?

    I use a bead homogenizer (Precellys 24) which can generate quite a bit of foaming. I am thinking of using anti-foam A (from Sigma) to reduce the foaming. The homogenate will be used for enzyme activity measurements and western blotting. Any comments welcome. Thanks.

    Jonathan Mark Wilson · University of Porto

    Thanks for the advise. I will see if I can get away with running a dot blot rather than the western. I was planning on measuring enzyme activity first and then the immunoblot on the same homogenate.

  • Mary A Lokuta added an answer:
    Can anyone recommend a protocol for western blot preparation of freshly isolated human neutrophils?

    I'm currently conducting research with human subjects and would like to analyze human neutrophils by Western Blot. We get 1-2 patients a week -- would it be possible to flash freeze the neutrophils and lyse them later, or is it better to prepare Western samples using fresh neutrophils?

    Mary A Lokuta · Stratatech Corporation

    I have always preferred an aggressive approach as I have found them to be somewhat resistant to lysing and chock full of proteases.  A good description can be found in my old JLB paper.  I have tried syringing instead of the alternate freeze/thaw described below but believe the syringing ruptures the nuclease and reduces the quality of the RNA.

    Good luck!

    "lyse in 50 mM HEPES, pH 7.6, 75 mM NaCl, 0.25% deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, 1 M NaF, 5 mM MgCl2 with protease inhibitor cocktail (P-8340, Sigma Chemical Co.), phosphatase-inhibitor cocktail
    (P-5725, Sigma Chemical Co.), 2 mM phenylmethylsulfonyl fluoride, 100
    M sodium orthovanadate, 900 M benzamidine, and 1 mM phenantroline.
    Samples were subjected to three alternating freeze/thaw cycles, and the debris

  • Al Muataz Yacoub added an answer:
    How to reduce multiple bands in western blotting?

    Hi, I am using monoclonal antibody against B-actin but I got multiple bands in that blot. I tried 1:100, 1:500, 1:1000, 1:2000 and 1:5000 primary antibody concentrations, but still same thing happens. I am using secondary antibody 1:5000 and 1:10000 conc. I am also used 5% BSA and 5% skimmed milk for blocking and for antibody incubation. I also tried by putting blot for overnight blocking. I am trying incubation of primary antibody for overnight at 4 degree Celsius and at 37 for 2 hrs,  still I got 4 bands instead of band of interest. I also tried multiple washing loke for 5-7 times. Please give me any suggestions. 

    Al Muataz Yacoub · Claude Bernard University Lyon 1

    Try to use new cell lysate, sometimes when you store the lysate at -20, if it s not enough to stop their degradation

    Also try higher dilution, for me i am using EF1 alpha and the recommended dilution is 1:1000, but i got multiple bands, so now i am using 1:10000 and it works great

  • Thanh N Nguyen added an answer:
    Why does my BN-PAGE run keep terminating prematurely? What can I do to fix this?

    yeah so i am running at constant voltage like 50V which gives me a current of upto 3mA but my run keeps terminating prematurely and i have to check on it every hour or so, each time i raise the voltage by 20V just to get it running again but the problem is i end up with frowning protein bands, like inverted smiles, not pretty at all... so i end up thinking the run was too rapid... i tried running at constant current but the bio-rad powerpack i am using will not go below 10mA when i set it manually and that shows a reading of like 500V!! i dont even want to imagine what sort of bands i will get at that. i see publications with really neat bands and i want bands like that, they use something like 30V for like 30 min then 80V for the rest of the run others use constant current something like 1.5mA for 15-17hrs all at 4 deg.C.

    Thanh N Nguyen · Monash University (Australia)


    If you already try to use the fresh cathodes (even fresh anode) and it does not help, it could be just simply changing your top chamber or power pack to see... Also make sure that there is no leakage of the cathode from the top chamber into the bottom part. Sometimes, a very slow unobvious leakage can cause that problem too... I do not think it's the buffer thing but rather the way things are set up.

  • CHANDRA SHEKHAR DASARI added an answer:
    MMP-2 bands are giving streak lines across the gel.
    I attached the zymogram image. The top line is MMP-9 giving distinct band. But the MMP2 band is like a straight line across the gel both latent and active forms even in the marker lane. Could any one suggest possible reasons for this pattern of zymogram.?
    CHANDRA SHEKHAR DASARI · Indian Institute of Chemical Technology

    Thats' a nice explanation. I also stick to that. These days, I am running gels in 4degrees. So that I am not getting the streak again.

  • Tamar Cohen added an answer:
    Where can I obtain a complete protocol for an insulin secretion assay in a MIN-6 cell line?

    Does anyone have a complete protocol for an insulin secretion assay in a MIN-6 cell line? I'd like to test the effect of our drug on insulin secretion and am still learning about cell culture. Also, is it possible to measure insulin secretion and to be able to use the very same cells to perform western blot or RT-PCR (to try to probe for the mechanism influencing the secretion)?

    Thank you so muc

    Tamar Cohen · University of Southern California

    Thank you so much, I'll try that paper.

    Ercument , it seems like the protocol would be helpful too. Do you mind sending it?

    Thanks again

  • Paul E Harris added an answer:
    If planning to change the thickness of Western Blot gels from 1.0 mm to 1.5 mm, how much more time should the SDS-PAGE and WB be operated?

    Hello everyone,

    Recently, I have been planning to switch the thickness of SDS-PAGE gels for Western Blot from 1.0 mm to 1.5 mm to accommodate for larger samples. I use Life Technologies' NuPage Tris-Bis 4-12% gels. If I had been running for 100 min at 70 V previously with 1.0 mm, how much longer (and perhaps with higher voltage) should the gels be run if the thickness is bumped up now to 1.5 mm? As for WB, I had been running for 60 min at 30 V. 

    The company's protocol does not say much about any modification regarding a change in the gel thickness.

    I am wondering if anyone had to make such a change as well and has already figured out how much more time relatively these two steps would have to be taken.


    Paul E Harris · Columbia University

    Regardless of any advice given here. You really should check the transfer of protein from gel to membrane using the prestained molecular weight standards. We too us the NuPage 4-12 gels and get good transfer of 20-30 ug  on a 1.0 mm Gel to nitrocellulose in 20-30 minutes and 45 minutes for the 1.5 mm gels..but it all depends on your blotting system.

  • Maria Teresa Borrello added an answer:
    I am trying to use western blots to detect methylated histones, but I'm getting really inconsistent results. Is anyone else trying to do this?
    I am using a non-Drosophila fly species and I can sometimes get good results with the control, non-modified, histone H3 antibody. But sometimes I don't get any band. I'm not sure if it's a problem with my sample preparation or with the transfer (could I be transferring the histone proteins through the membrane?) I would like to get the insight of someone who has experience with trying to detect histone proteins with western blots.
    Maria Teresa Borrello · University of East Anglia

    I have been experimenting the same as you and still does not seem to be good enough.. Julie, did you succeed? I have all the time variable results using THP-1 25x104 cells in one mL re-suspending the pellet in 50uL of lysis buffer and loading 10ug of protein per well. Sometimes it work, sometimes not...The most frequent problem is that I do not have bands in the desired region. At the moment we just have precast gels at 12%. We are a medicinal chemistry lab and I am not very familiar with biological techniques. ANy suggestion will be much appreciated.

  • Dieynaba Ndiaye added an answer:
    Is anyone doing western blot for measuring 3BHSD, STAR and SMA?

    I am going to do in vitro culture of bovine luteal cells. I want to measure these proteins by western blot but I have have protocol belonging these proteins:

    1- 3 B hydroxysyeroid dehydrogeanse

    2- STAR( steroidogenis acute regulatory protein)

    3- smooth muscle actine

    Dieynaba Ndiaye · INRS

    Anti StAR and anti HSD3B from abcam works well for me.

    My concern is for an antibody able to discriminate between Cyp11 and Cyp17. and it's not that simple when you know a lot of informations are confidential...

  • Rohail Jawed added an answer:
    I am unable to get the band on Western blotting but the protein is highly positive on ELISA, what could be the reason?

    I am working with 4 pure mitochondrial enzymes without any tag protein whenever I used this enzyme on ELISA I got the highly positive signals but when I do western blotting only 2  proteins give me bands but the remaining 2 proteins I can't see. Please explain what could be the reason of this issue. Thank you.

    Rohail Jawed · University of Karachi

    Thanks for your answers

  • Joseph R Casey added an answer:
    How can I store samples for western blotting?

    Which is the most efficient way to store samples for general western blotting (not phospho) for weeks, as cell pellet at -20°C or in RIPA/lysis buffer after lysis or protein with SDS sample buffer at -20°C or on the membrane or may be something else anyone can suggest? Thank you.

    Joseph R Casey · University of Alberta

    You can also store blots on which protein has already been transferred. Blots, once dried, can be kept in sealed containers, sitting on paper tissue, in a refrigerator for long periods of time before probing with primary antibody.

  • John C Schmitz added an answer:
    What is the best method to normalize between gels?

    Hi, I'm running Western blots, looking at various proteins involved in the mTOR pathway and I'm looking at phosphorylated versus total protein ratio.

    I have 8 replications (animals) and 4 treatments and am using gels with 15 wells so I can only load 3 reps (each with their 4 treatments) per gel.  I'm using the same procedures but sometimes exposure times will differ somewhat on each occasion.  
    What would be a good way to normalize between different gels? How can I account for differences between gels. 

    I was also wondering if anyone ever uses a positive control and normalizes to that.  Right now my data is organized as phosphorylated protein as a ratio of the positive control (which is a pooled random muscle sample), total expressed as a ratio of the positive control  and then the final value is a ratio of those ratios as in Phosphorylated versus Total.  

    John C Schmitz · University of Pittsburgh

    Another option is to use both antibodies (phospho and total) on the same blot. The antibodies need to be derived from different species (a rabbit antibody and a mouse antibody). The secondary antibodies contain infrared dyes that are detected at different wavelengths. We use the Infrared scanner from LICOR to detect both signals simultaneously (many core facilities or big labs have one of these scanners). The primary antibodies must also recognize different protein epitopes (to avoid interference). Admittedly, this takes some trouble shooting to determine the optimal conditions, but once the conditions are set, the technique eliminates normalization between two gels. We typically test for GAPDH just to make sure our protein concentrations were similar but we don't normalize to GAPDH. Just the ratio of phospho/total protein.

    Good luck!

  • Frozan Safi added an answer:
    If I reprobe a membrane that I previously developed using anti-mouse with anti-rabbit, why do I still see the signal for the mouse antibody?

    If I reprobe a membrane that I previously developed using anti-mouse (without stripping) with anti-rabbit, why do i still see the signal for the mouse antibody? My colleague in the lab does not have this problem and we use the same antibodies, dilutions and incubation times. I am trying to see two bands on one membrane that are of different sizes. I don't know why he does not see the old bands but I do. 

    Frozan Safi · SickKids

    Thanks Mark Livingstone, indeed it turns out it that all his primary antibodies contain sodium azide which is why he does not see the signal from before even without stripping the blot. 

About Western Blot

Western blot is a widely accepted analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide. The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are stained with antibodies specific to the target protein.

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