Western Blot

Western Blot

  • Maritza Kruger added an answer:
    How is the better way to normalize phosphorylated proteins analyzed by Western blot? With structural proteins or with their total protein pair?

    I am working with phosphorylated proteins, using the western blotting technique. I have seen in the literature both phosphorylated proteins normalized with structural proteins, such as actin, or tubulin and with their total pair (phospho-AKT/AKT/ actin), and only normalized with its total pair, such as phospho-AKT/AKT. What is the better way to do this?

    Thank you for your atention.

    Maritza Kruger · Stellenbosch University

    I agree. Normalise to total Akt and then also a loading control such as actin. 

  • Lei Wang added an answer:
    What is the best way to detect extracellular proteins using western blot?

    I'm working with a neuron cell line in culture and I will want to check the levels of different extracellular proteins using western blot

    Lei Wang · Johns Hopkins Medicine

    As people suggested already, try ELISA first.

  • Steingrimur Stefansson added an answer:
    What are some good housekeeping proteins to use for Western blot for inflammatory diseases?

    I'm working on Western blotting for an inflammatory disease and I'm having trouble finding a good loading control to use. I have previously tried beta actin, GAPDH, and alpha tubulin, but all appear to be too saturated. TBP was then tested but the variation between individual samples is too high. I'm thinking about trying ywhaz next. Anyone have any other suggestions? Thank you lots.

    Steingrimur Stefansson · HeMemics Biotechnologies Inc

    Hi Cassidy, what is your inflammatory disease model?

    Is it in vitro or in vivo?

  • Anne von Koschembahr added an answer:
    Where can I find Phospho atm protein in western blot in MCF 7 cells?

    Can I get it in MCF 7 cells or should I expose my mcf7 cells to UV to get this protein? Can you please suggest me something? How much of UV should I expose to the cells to get phospho atm? This protein will express when there is a break in double strand of DNA.

    Anne von Koschembahr · University of Cincinnati

    The level of phospho ATM may be low in your cell line, so I would recommend that you use UV or another DNA damaging agent (IR, for example) that is known to induce ds DNA breaks. I don't know what wavelength of UV you plan to use, but a higher dose will ensure that you have a sufficient amount of damage to activate this signaling pathway. For example, we can detect increases in phospho-ATM when we UVB irradiate primary human skin cells with a minimal dose 90 mJ/cm2. Then run both control and cell stressed lysates and that will help you to determine if ATM is activated under baseline conditions. You can validate in a third way by purchasing cell lysates (such as Cell Signaling Technology) that will serve a positive control for pathway activation.

  • Isabelle St-Amour added an answer:
    Can I re-use a primary antibody after a western blot?

    I am currently using skimmed milk for blocking.

    My antibodies (anti-phospho antibodies against proteins of insulin signalling pathway) are quite expensive.

    Please, suggest methods for reusing and storage of the antibody.

    Isabelle St-Amour · Laval University

    You can definitely re-use the primary antibody solution either by adding NaAzide to the solution and keeping it at 4°C or by freezing it at -20°C. Most antibodies can support up to 4-5 freeze-thaw cycle so it is best to keep track of this. However, you will see that some antibodies only work once.

  • Abdul Mannan added an answer:
    Can anyone help with Western Blot analysis?

    I am doing western blot and the problem that I encounter is that the target protein and housekeeping proteins' molecular weight is close enough to cut the gel to probe on separate membranes. However, they get separate enough to distinguish between two easially.

    My question is after probing with one ab, do I need to strip before probing with ab for other protein? 

    Thanks all of you!

  • Marziea Rezaei added an answer:
    Can any one help me about western background?

    I'm doing  2D with protein extract from cell lines (on 11cm Strip,12-14h rehydration and  27000 vh IEF) and then transfer it on PVDF (with semi-dry, 22v, 40min), next blocking in 5% skim milk in PBS for overnight 4C , then washing with PBS-T and incubation with human serum (1/100 dilution) for 5h, after that washing and incubation with secondary anti-human IgG (1/2000 and 1/4000) for 1:30 h and finally exposure with ECL(Bio-rad) for 5min, dry it with paper towels and exposed to X-ray film.

    with this process I have two problems,

    1, strips: after IEF step, seems in the middle of strips dosnt any gel and at the ends of it, gels swollen. Do you think it is for over focusing?

    2, background: i have an unusual cloudy background on both sides of the membrane toward the middle of it (I should note that I also have this problem with one-dimensional Westerns and mAb), I think this is not due to of incomplete blocking. would you please get me your idea?

    Marziea Rezaei · Shiraz University of Medical Sciences

    Dear alls

    thank you for your coments

    i can resolve my problem

    my protocol is<

    blocking with 5% skim milk, over night 4C

    primary antibody(human serum), over night 4C

    Secondary antibody (1/30000 dilution), 1h 

    between each steps, washing with TBS contain 0.1% tween 20 6times

  • Edmund Pool added an answer:
    How do you prevent PBMC protein degradation?

    After my western blotting for TBP in PBMC, I saw no bands for 2 samples which may be due to protein degradation. I have kept the two samples for 4 months in -20C with protease inhibitor cocktail (Roche). Any suggestions? Thanks very much.

    Edmund Pool · University of the Western Cape

    Agree with Steingrimur - always store your samples in small aliquots at -80.  You can also heat denature aliquots prior to storage at -80C. Will kill off proteases.

  • Mónica Suárez Korsnes added an answer:
    Should I use phospho-AKT Ser473 or Thr308 antibody in Western blot?

    I want to look at the phosphorylation of AKT in HUVECs treated with hyperglycaemia. Can anyone suggest whether I should use the pAKT Ser473 or Thr308 antibody? Does it matter which one of the phosphorylated isoforms I use? Thanks

  • Xin Jiang added an answer:
    In you experience with Li-COR system for quantative western blot, what's the level of "bleed through" you have b/w two color channels?

    How does that compare to your expectation? On a related note, is the overall accuracy of quantitation higher with Li-COR/FL than blot-strip-blot with CL? Many thanks!

    Xin Jiang · University of Washington Seattle

    This is great, thanks again Stephan!

  • Zuzanna Michalak added an answer:
    Western blot for +200 kD transmembrane protein is not working any more?

    I am running western blot to detect transmembrane protein of >200 kDa (SCN1a). I extracted my Proteins with 5M Urea Buffer, and I keep them at 4*C. I run Western using Tris-Acetate gradient gels 3-8 % with Tris acetate running buffer (Invitrogen). I prepare my samples in Laemli buffer (BioRad) or Sample buffer (from Invitrogen) with BetaMercapto or LDS as reducing agents. I do not boil the samples. I use wet transfer for 2h at 30V.

    The westerns worked very well, and suddenly from one day to another it stooped. First progressively my high MW band that before were very strong after 5 min (in the cassette) started to fade, then my Low MW, and by the end using the same samples I was unable to detect any band after few hours!

    Please give me any possibilities what the problem is. I tried to resolve the technical problem by changing the lot of gels, running buffers, Laemli, secondary and primary antibodies, electrophoresis and transfer systems...

    And the last test my friend performed for me using hers and mine samples, and all samples looked ok on PonceauS but the blot worked only on her samples - meaning there is something wrong with mine!

    I need to add that these samples are VERY important and impossible to reproduce (as it is a human material).

    I will be very thankful for any advices!



    Zuzanna Michalak · University College London

    Antibody is against Peptide (C)TASEHSREPSAAGRLSD, corresponding to amino acid residues 465-481 of rat Nav1.1 (Accession P04774). Intracellular loop between domains I and II (Alomone Labs, ASC-001). The problem is that I use actin and NeuN as controls, and even these antibodies are not detected!

  • Michael Chinkers added an answer:
    Is a polyclonal antibody acceptable to use for detecting phosphorylation level at ser 78/82 residue by western blotting?

    I am planning to use phospho 78/82 HSP27 antibody to detect phosphorylation level at ser 78/82 residue by western blotting. I found an antibody but it is polyclonal (company is bioworld technology inc.), so is it a problem for my experiment?

    Michael Chinkers · University of South Alabama

    Very acceptable. It's a lot easier to generate high-affinity polyclonal antibodies which, by virtue of that affinity, tend to give a higher signal-to-noise ratio than most monoclonal antibodies. This is particularly true in this case due to the affinity purification of the polyclonal antibodies you plan to use.

    The usual practice would be to generate polyclonal antibodies to conjugated phosphopeptides and affinity-purify them on a matrix containing the phosphopeptide of interest, as well as removing antibodies that bind to unphosphorylated peptide. I've had very strong clean signals in Western blots with rabbit phospho-Smad antibodies from Cell Signaling.

  • Ahmed Y Shanab added an answer:
    How can I prove caspase-independent cell death?

    My compound induced apoptosis proved by annexin v staining, in the case of western blot experiment I was trying to prove through caspase and PARP cleavage. I could not absorb caspase-3, 8 & 9 cleavage, interestingly PARP was disappeared (no cleavage).

    How can I prove this kind of cell death, which mechanism will be involved?

    Ahmed Y Shanab · University of Georgia

    Check other capsase dependent proteins as Fodrin (alpha-spectrin) by wester blot... annexin-v mostly indicated necrosis rather than apoptosis.... however, the caspase kit assay may help as well.

  • Olivia Ihedioha added an answer:
    Why am I getting a smeared molecular weight marker in Western blot?

    After running SDS gel and transferring a protein of 65kDa to PVDF membrane in a Western blot, I observed that the molecular weight marker appeared smeared. I used 10% separating gel and 4% stacking gel, I made a new APC and a new running and transfer buffer but still couldn't figure it out. This is the second time it has happened recently. I have done a series of Western blot in the past few months without any challenge. 

    Please can someone help me.

    Olivia Ihedioha · University of Manitoba

    Actually I don't think 10 minutes is necessary, but that has been the protocol we followed in our lab, I guess it isn't working for me. I need to change it base on the suggestions from Karoline and T.K concerning soaking PVDF for few seconds.

  • Carlos G Perez-Garcia added an answer:
    Why in western blot, every protein that I have checked appears in the same molecular weight?

    I have checked Cg A, Vmat 2 AQP4 from CSF.. and all of them appears at 50KD. I don't know what is going on.

    Carlos G Perez-Garcia · Salk Institute

    Hi Leandro, you are probably detecting non-specific bands, you can start by changing the dilution of your secondary antibody, also use washing with TBST and either BSA or powder milk, that one works fine for me. A more suitable option is to use a light chain specific antibody to avoid detection of the heavy chain ~ 50 KDa that always give you a lot of noise. Goodluck. Saluda a Agustin de mi parte, un saludo , Carlos

  • Marcin Nowicki added an answer:
    My protein is not getting expressed in E.coli. How can I overcome this problem?

    I'm trying to express a mammalian protein in E.coli. The protein is cloned in pET-Sumo vector and is 55 kDa in size . I tried cell strains like Rosetta DE3 and origami but the protein failed to express in both cell lysates and pellet(verified by Western Blot). The sequence is in-frame and induction was checked at different IPTG conc. with different temperatures. How can I overcome this problem?

    Marcin Nowicki · Research Institute of Horticulture in Skierniewice


    Based on all the info in this thread, the likely suspects are in-frame cloning and/or codon usage. If it keeps not working @E.coli, you could always transform yeast or Agro-transform some plants. Good luck!

  • Ketil winther Pedersen added an answer:
    Why am I getting an odd CD63 Western Blot result?


    I am doing Western blots for exosomal CD63. When I run the blot (under native denaturing conditions) my CD 63 appears to be around 200 kd. This cannot be!

    Can anyone help? I used a Santa Cruz antibody and the data sheet says CD63 should appear at 26 kd when it is not glycosylated.


    Ketil winther Pedersen · ThermoFischer Scientific,

    We also observed a smeared pattern for CD63 (40-65 kDa) both from cells and exosomes (Oskvold et al 2014) using the clone TS63 when we compared the profile in different B-cell lymphoma derived exosomes. It also appeared to be different smeared pattern depending on the origin.

  • Liu Liam added an answer:
    How could I get a sharp band when doing WB?

    I've done Western Blotting using total Akt, pAkt ser 473 and pAkt thr 308. For total Akt and pAkt ser 473, I mostly see a very clear and sharp band. But there are so many non-specific bands for pAkt thr 308. All of the conditions are the same. I use 5% non-fat milk to block and 5% BSA to dilute the primary antibody. 

    Thank you!

    Liu Liam · Chinese Academy of Sciences

    Maybe the buffer for primary antibody, espercially the pH have something to do with the perfomance of your antibody, you can try to change it. Generally We use TBST (0.5% tween 20) to wash the membrane.

  • Baylin Bennett added an answer:
    Has anyone used the ProteinSimple Wes capillary western blot system?

    Does it work as well as advertised? How does it compare to traditional westerns?

    Baylin Bennett · Oregon Health and Science University


    Yes I went with two other coworkers from my lab to the colleague's lab, whom I previously mentioned, aboout 3-4 weeks ago. The two salesmen met us there, as well as one other scientist who was interested in Wes. My coworkers would agree with my experience. 

  • Ricardo Bonfim Silva added an answer:
    Can we keep the cell culture flask for some time after the treatment time is over for the purpose of protein isolation?

    I have to do a western blotting for observing the expression of specific protein of interest. Cells were treated for a specific time with a specific substance and incubation time is over. Now can we keep the cells at 4 c or at -20 for some time without having any false effect on the cell line.

    Ricardo Bonfim Silva · Indiana University-Purdue University Indianapolis

    if you gonna process the samples soon you can freeze at -80, don't need put in liq N2. And very importante... after take out the cells from culture wash the pellet at least two times with PBS because the serum from medium interferes in the analysis. After washing, aspirate the PBS and freeze just the pellet.

  • Saleh Alkarim added an answer:
    Can anyone who has experience with western blot of AHRR suggest methods to obtain the signal band?

    I have been working with western blot analysis of AHRR for months, but I have never seen signal band of AHRR. However, I can see actin bands. I use proteins from cell lysis from HepG2 and Hepa 1c1c7. And I load 20 ug protein per lane. The primary antibody for AHRR is 1:500, and second antibody is 1:25000. Thank you.

    Saleh Alkarim · King Abdulaziz University


    i think this difference In expressing due to the type of cells , Where i read the article discussed this issue and showed that the AHRR low endogenous expression in normal HMEC with relatively The resulted increase expression and activity correlated with the development of cellular malignant phenotypes, most significantly epithelial-to-mesenchymal transition.

    you can see the attachment in this link , and pleas read the PDF , i hope help you.

    Good luck

  • Sebastian Marwitz added an answer:
    Why does the molecular size of a GFP-tagged receptor exceed the expected size on a western blot?

    Dear all,

    We tagged a chemokine receptor with GFP and expressed this in an epithelial cell line to analyze receptor trafficking. This works good, the receptor is expressed, we do see receptor internalization and ATP changes after challenging. We also used this receptor for co-IP of a new ligand. Also the co-IP works fine as we could detect the new ligand after fishing using anti-GFP beads. However, we do have a problem with the molecular size of the receptor. The receptor itself has molecular size of 43 kD, with GFP we expect a size of 69 kD, however, what we see is a size of approximately 100 kD, sometimes even higher. Why? Any explanation? We checked the plasmid for orientation and that it contains only one GFP. Thus, I do not believe that we have a construct with two GFP which would also add up to 100 (receptor: 42 + GFP: 28,5 x 2 = 99). Any other explanations?

  • Khuong Le added an answer:
    Does anyone have set hands in purifying the his tagged recom protein (secretory) NGF?

    I'm working on Novel protein therapeutics for peripheral nerve regeneration. My protein NGF (Nerve Growth Factor) was expressed in Sf9 cells and secretory protein was unable to purify under native or denature conditions. I have performed Ni-NTA agarose resin and followed by silver stain and western blot. Other side I also tried to purify using Q-Sepharose ion exchange chromatography followed by Ni-NTA agarose, but no change in purity.

    Please advise if any one has done earlier

    Thank you

    T M P RAJU

    Khuong Le · Hôpital St-Boniface Hospital

    Is your initial binding buffer for Ni2+ column is at pH 8.0? Try pH 8.0 for all the step from cell lysis to the elution step. Good luck.

  • Jan Adrianus Veenstra added an answer:
    How to use a Western Blotting specific antibody for IHC (for PFA fixed & paraffin Embedded tissue)?

    I have an antibody in my lab specefic for western blotting only, but I need to detect same antigen in a PFA fixed tissue. I tried to retrieve epitopes using Citrate buffer, HCL treatment & Proteinase K, but it's not working with any of them. Can any one help me in getting the signals in IHC. 

    Jan Adrianus Veenstra · Université Bordeaux 1

    Here is yet another thing to look at. Does the antigen have a lysine residue ? This is usually highly immunoreactive, meaning the antibodies will recognize this sequence, but unfortunately, the PAF will react with the lysine and your epitope is gone. In principle a fixative that does not attack primary amines might work, but those are often not very good.

  • Marzieh Eghtedardoost asked a question:
    Could I use EnVision™ Detection System from dako ( e.g. K5007) for western blotting technique?

    Could I use EnVision™ Detection System from dako ( e.g. K5007) for western blotting technique? or it is only for IHC?

    Best regards

  • Alexandra Müller added an answer:
    What are the causes of a bad Western blot result?


    I did the Ponceau on the last run, and it came out looking like these two images, attached.

    What could cause this, besides bad gel?


    Alexandra Müller · Ruhr-Universität Bochum

    You can get rid of DNA by sonicating the samples after adding SDS sample buffer and boiling. A sanitation bath is best-suited for this purpose. Sometimes it helps to reduce the protein concentration by adding more 1x loading buffer and then loading twice the amount of sample to each pocket. Also additional boiling/vortexing circles may help.

  • Lalita Sharma added an answer:
    How can I get bands for HDAC1 in western blotting?

    I did Western blotting for HDAC1 (1:1000 dilution of antibody from cell signaling). I got v. faint band after 1 hour exposure. I stripped the membrane and increased the dilution to 1:200 and this time I did not get any band. What can be the reason and how can I get proper bands for it. Is HDAC1 comes only in fresh membrane or it comes with stripped membrane too?

    Lalita Sharma · Jawaharlal Nehru University

    Thank you so much for this elaborated protocol and a valuable suggestion. I will follow the same and hope this time will get band . we stored the antibody in - 20 degree since then and it was thawed only few times. The protocol I mentioned was the protocol  after which I got that faint signal. Now I will follow the exact protocol as of yours and yes , the cell line also matters, may be my cell line (DU145) expresses it at lower level.That's why I am getting the faint band. 

  • Jürgen Denecke added an answer:
    What might be the reason for the bad sample migration in my western blot?

    Recently I tried a western blot on various human muscle samples. Unfortunately, some of the samples migrated like the fourth sample you see in the picture. It seems like the proteins in the sample are too aggregated, but I really don't know what to do to "unpack" them and no one in my lab can give me any advice about it. I used RIPA lysis buffer to lyse the muscle sections, and broke them down by freezing and thawing them a few times.

    Any advice would be immensely appreciated.

    Jürgen Denecke · University of Leeds

    When you make protein extracts from different tissues, you should first normalise them by total protein concentration (before adding the sample buffer). Invariantly, when using manual grinding, or even freezing/thawing, the extraction efficiency is not the same, so you need to quantify total protein levels in your various samples. Afterwards, you bring all concentrations down to the lowest in your series by appropriate dilutions. Then you add equal volume of diluted samples and sample buffer, boil and run the SDS gel for later Western analysis. This means, when you run the SDS PAGE, you load equal voilumes and equal protein concentrations in each lane. If you just measure protein concentrations, and load different volumes to achive the same amount of protein in each lane, the samples will not run well. Always equal volumes and equal protein levels, it's more work, but it is necessary.

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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