Western Blot

Western Blot

  • Thibault Bouderlique added an answer:
    Why I get a blank signal on western blot?


    I am new to western blotting and so facing the problem of blank blots.

    i want to assess the expression of two membrane bound proteins, 45 & 74 KD, in rat heart tissue. My tissue samples are stored in liquid nitrogen.

    My protocol as follows:

    Grinding 60-80 mg of tissue in liquid nitrogen using mortar and pestle

    incubation of tissue powder in RIPA buffer + protease and phosphatase inhibitor cocktail for 1 hour at 4 C, with needle shearing using 21 guage needle.

    centrifuge at 22,000 xg for 20 min, collect the supernatent and discard the pellet.

    Denaturation of proteins was done using 2x lamaelli buffer and boiling at 95 C for 5 min.

    loading 40 ug / well of the total protein and using 10% SDS-PAGE.

    transfer of protein to nitrocellulose membrane, and assaessing effiency of transfer using ponceau s stain

    blocking membrane in 5% non fat dry milk, overnight

    incubation with primary antibody, many different dilutions, overnight at 4 C

    incubation with secondary antibody for 2 hours at room temp.

    colorimetric detection using TMB substrate solution.

    after this, I always gt a blank signal with my two membrane proteins.

    noteworthy,  I had a signal when I probed the membrane with actin antibody. but this signal washed away in the substrate solution with continuous shaking, leaving also the membrane almost blank, and turning the color of the TMB substrate sol. into light blue

    My primary antibodies does fine in IHC, so I do not think that they are blamed

    My secondary antibody gives a deep blue color when I incubated it with TMB sol. in an eppendorf.

    I am not sure whether the problem is in my protein extraction protocol or in one of my antibodies, including my secondry, or in the TMB substrate sol.?

    Thibault Bouderlique

    Actually, the fact that an antibody works in IHC doesn't mean that it will do for WB.

    The proteins are denatured by the b-mercapto ethanol and thus an antibody that detects a ternary or quaternary strucutre in IHC won't be able to detect anything in WB.

    As you can detect actin, it seems that you have proteins on your membrane.

    I thus strongly advice you to use a positive control (tissue known to have big amounts of your protein of interest, or some recombinant protein), that would allow you to see if your antibodies are the proper ones for your application.

  • Raj Kumar added an answer:
    Can you show the picture of your worst Western blot, and explain the reasons for this disaster please?
    I start this topic in order to collect the gallery of the photos of Western-blots (WB) as ugly as possible. The detailed description of the problem and the technical solutions were applied to resolve it are strongly suggested, however ugly WBs without the clue what is wrong are welcome to discuss it here. The overall idea of this post is to help the young researchers to resolve the problem with simple and cheap tricks. On the photo below you can see the lightning in the middle, dust in the upper left corner, small air bubble in ECL solution in the upper right corner. The background is not fully grey, means ECL was not properly distributed, only band visible is not specific binding of the antibody to the slightly degraded marker band (right).
    Raj Kumar

    Dear,  Prem Subramaniam  yes the fat band is 14kDa, The exposure of B-Actin is more then the lc3. Yes i am sure the fainter band is lc3-II. Actually the marker is also smeared .

  • Bassam Alkotaini added an answer:
    How to measure the protein concentration of a sample containing Laemmli buffer?
    I am very new to Western Blot (WB). Before starting WB, I wanted to know the protein concentration of my sample. So, I tried to do it with a microplate reader at 595nm with Bradford Reagent (Bio Rad company). I diluted my standard (1mg/ml) with 2X laemmli buffer (diluted 1/50 times) to get different concentrations of standard ranging from 40ug/ml to 0.25ug/ml. Then from this mixture, I added 160 ul to each well, then 40 ul of dye to make up the final volume 200ul. But the absorbance is very high and inconsistent. Even the blank shows very high absorbance. How can I sort this out? If anyone has a nice protocol for this please do help me.
    Bassam Alkotaini

    As i know, laemmli buffer contains comassie blue which binds to proteins. Bradford assay will not give accurate results cause comassie adsorbs around 580 nm. I advise you to prepare your sample  in other buffer 

  • Hong-su Park added an answer:
    Standardizing supernatant?
    I'm collecting supernatant as samples and measuring certain proteins released by cells/tissues after treatment. I would appreciate suggestions on how to standardize supernatant in running Westerns and ELISA. I am aware that running these assays using LYSATES we use total protein content measured by BCA assay or other protein assays. But what about if you are using supernatants where we don't expect intracellular proteins to be present? What should be used as a measure of uniformity in loading the wells?
    Hong-su Park

    When I see papers showing WB results to detect mature IL-1b or active caspase-1 from supernatants, they did not show any loading control. It is assumed that precipitated proteins for a sample are derived from the same volume of supernatant with the same number of cells as other samples. So without any loading control, the data is acceptable, I think.

    The following paper saying that IL-1b release is accompanied by other cytosolic proteins, shows that upon necrosis, depending on the treatment, some intracellular proteins are released into supernatants.

    In the Fig. S4 (A), for the WB with both cells and supernatant, they seemed to use the same control which is consistent among samples. But unfortunately, they did not indicate which protein it is.

    And if you see other figures, depending on the cell line and different treatments, they also detected actin from supernatants (even at 0 hour post-treatment with LPS).

    If your treatment does not induce much necrosis, you may consider trying to detect one of those intracellular proteins to check whether a consistent amount is released.

    • Source
      [Show abstract] [Hide abstract]
      ABSTRACT: The NLRP3 inflammasome is involved in caspase-1-dependent maturation of IL-1β in many contexts. A two-signal model has emerged for IL-1β maturation, with LPS providing "signal I" and diverse agents such as ATP, Nigericin, streptolysin O, uric acid crystals, and alum salts capable of acting as "signal II." In the absence of signal II, pro-IL-1β is upregulated but typically fails to be processed or released. What unites signal II stimuli has been debated, with the ability to promote K+ efflux suggested as a common factor, but the mechanism of IL-1β release remains unclear. Here, we show that all examined inflammasome signal II agents triggered necrosis, which was highly correlated with their ability to promote IL-1β release. IL-1β secretion occurred in tandem with the release of many additional proteins and was confined to necrotic cells. Thus, signal II agents initiate inflammation by promoting necrosis-driven IL-1β release, suggesting that IL-1β represents an inducible danger signal. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
      Cell Reports 05/2015; 11(10). DOI:10.1016/j.celrep.2015.05.003
  • Amritlal Mandal added an answer:
    Does anyone have the experience with mouse DRG neuron culture (for western blot analyses)?

    I have a protocol to dissociate cells but I don't know how and at which density the cells should be plated to perform western blot analyses.

    Amritlal Mandal

    Try seeding 10^4 neurons/cm^2. This should give you optimum cell growth and protein expression for WB analysis.

  • Bernard J Moncla added an answer:
    I have problem in western blot, can anyone help?
    • When I prepared my western blot for Protein of interesting and B actin loading  control .
    • I have get result with b actin blot but my protein intersing  blot no band ,,
    • protein concentration 25 microgram /microliter,
    • I prepared 4% gel SDS PAGE
    • blocked for 1 hour 5 % milk in TSBT
    • primary overnight
    • secondary 2 hours
    • wash step 3 time \15 min
    Bernard J Moncla

    I think Vakamullu is correct, start with the basics.  Have you performed a "checker board"? It maybe you need to start by getting your system to work with a better known antigen antibody combination.

  • Sesha Kiran Kollipara added an answer:
    Why does my SDS-PAGE shrink when I try to cast it?
    I am attempting to cast either 10% or 12% gels for a western blot, but every time I attempt to do so, the gel shrinks at least a centimeter from where I originally filled it to to the point where the gel is too short to work with. I am using a standard gel recipe and once I cast the gel to the desired height, I add water to the top of the gel to help it polymerize and to smoothen out its top. As soon as I remove the water, it is very apparent the gel shrank. I leak test the apparatus with water for ten minutes before I cast so I don't think it is a leaking issue. Does anyone have any insight?
    Sesha Kiran Kollipara

    Its given clearly in Molecular Cloning Manual the amount of each component to be added for different concentrations of SDS PAGE Gel. What do you mean by optimising the addition of TEMED? He can check if the TEMED he is using old one or replacing with a brand new TEMED for formation of gel. 

  • Joydeep Bhadury added an answer:
    Can I use higher concentration of skim milk for western blot blocking?

    I usually do my blocking for 1 hour at RT using 5% skim milk, but now I am doing it already for 2 hours or so because the background is not clean with just an hour blocking. My skim milk powder is somewhat old, we bought it last 2011. Is it possible to increase the concentration of the powder or should I just buy a new one.

  • Kübra Gülmez added an answer:
    How can the protein detection range differ from proteins own molecular weight?

    According to UniProt, Mw of MeCP2 protein is ~53 kDa. However the antibody detects at ~72 kDa. Is it because of the secondary structure that the protein is not detected in its own mw range?

    thanks a lot!

    Kübra Gülmez

    Thank you very much for your help and answers!

  • Shreyasi Gupta added an answer:
    Can I use multiple primary antibodies (from same specie) simultaneously within one buffer during western detection?

    I wonder if multiple antigen detection using 2 or more primary antibodies (from same host) simultaneously in same buffer is possible? If not whether it is due to antibodies from same specie or using 2 or more together at same time in one solution? Your suggestions are highly appreciated!

    Shreyasi Gupta

    Choose antibodies which give bands not very close.like if protein A is 68 da then another either above 80 or below 40

  • Nur Basirah Ghazali added an answer:
    Why is the band not equal for my western blot result?

    Dear all,

    Today, I have problem with my western blot work. Actually, I have four experimental groups (wild type and KO mice model). From all those group I do protein isolation using RIPA lysis buffer to collect total protein from liver samples. Unfortunately, only from wild type mice with High Fat Diet treatment, I always cannot get good/equal band while for the other groups are ver nice. Little bit so confuse why always only in this group?even I already check the total protein concentration (BCA Thermoscientific) and use BSA 2 mg/mL as the higher standard value for my samples, looklike so weird why still give me the feature so many times?According to total protein concentration value, I think the calculation for all samples are correct, but need your helps what should I do know?I am stag now with my research and feel confuse with this situation.

    Please help me..Thanks a bunch.

    Best Regards,


    Nur Basirah Ghazali

    Hi Hendra. I want to make a point on the protein measurement. I use the same kit to measure my protein samples. I usually use 1mg/ml as my highest standard as according to beer-lambert law, any value greater than 1mg/ml usually will produce deviation in standard curve, hence the conc cannot be extrapolated from the standard curve when the absorbance of the samples is higher than the absorbance of the highest standard. So MAYBE your estimation could be wrong because of this. I suggest you dilute your samples, to ensure that the values are below 1mg/ml. Hope this helps!

  • Kaan Adacan added an answer:
    Why are bands are not visible in PVDF membrane after being stained with ponceau?
    I have transferred gel in PVDF membrane and stained with ponceau but I couldn't see any bands on the membrane, instead, the whole PVDF membrane is turned into pink color and the color is not going after prolonged wash also. I have used 30 microgram of protein and I transferred in semidry transfer apparatus. I have used the nitrocellulose membrane before that for the same concentration of protein and I could see the bands after stained with ponceau. I got this problem only with PVDF membrane. I have also activated the PVDF with methanol after transfer and stained with ponceau even if I couldn't find any bands. What might be the reason and how to see the bands and how to remove the background pink color?
    Kaan Adacan

    i used to activate PVDF membrane with %100 methanol for like 30sec to 1 min. After that i incubate them in the transfer buffer(%15 MetOH) atleast 5 to 10 mins. as you mentioned, i also use semidry. I incubate pack/paper  and the gel itself in the transfer buffer too. when it comes to transfer, i use 25V 2.5A and transfer time depends on your PVDF membrane there are differences between 0,45 and 0,25. You should be able to see bands after this protocol as i saw it before. You may want to stain your gel with commassie blue to make sure the gel itself does not contain more protein than it should have. I also saw people who stained their gel with commassie blue and washed with destain buffer. And they were able to make a retransfer from stained gel and got actual results from it. 

    And your second q. Most of the people will advise you to wash your membrane with tbs, pbs or tbs-t to remove pink background. I actually advice you to wash it with pure water ( ddH2O ). It will clear the background less than a minute.

    After this protocol if you wont be able to see any bands in your membrane ;

    - Your transfer time and V/A may cause problems with your membrane.

    - Your ponceau red ( make sure you add the acid fresh )

    - Your mambrane did not activated (?)

    -in some conditions, depends on your transfer time you mambrane can dry out and your proteins cannot be able to transfer to the membrane (semidry).

  • Anbalagan Moorthy added an answer:
    Do you snap freeze your cells and make cell lysates later to check phospho-proteins by western blot?

    I am working with leukemia/floating cells. Typically, I spin down the cells, wash them with ice-cold PBS, spin again and then lyse the cells immediately.

    I am thinking If you could snap freeze the cells after washing with PBS, then store the samples at -80C until you collect a batch of them and make cell lysates at the same time. One concern is that would this processing affect the phosphorylation status of your proteins of interest?

    I think it should not be a problem, while others hold different opinions. Please let me know your opinions. Thanks a lot.

    Anbalagan Moorthy

    Hi I always start with equal number of cells. between the controls and then I lyse the cells as soon as the treatment is over with SDS-loading dye and load equal volume of lysate. For example for a 6 well plate, I wash the adherent cells with PBS and then add 200ul of 1 X SDS-loading dye and lyse the cells, the lysate will turn viscous, we sonicate the lysate to decrease the viscosity and load 50 ul on gel to detect MAPK phosphorylations. Good luck.  

  • Temitope Adelusi added an answer:
    No consistency result in western blotting?

    I have been doing western blotting for several months and i use the same regents all the time but my western blotting results are inconsistent. I don't know why. Can anyone give me some suggestion ? i haven't treated the sample with any thing. i am just a beginner but going through lots of difficultly in western blotting. Everybody  get the result easily with the same protocol but its difficult for me . Please find the attachment of the my results (picture of western blotting) i think i have some  problem with the preparation of the cell lysate preparation .

    Temitope Adelusi

    WB could be very difficult to troubleshoot and when you get to the level whereby you are able to figure out the reasons why you have the result you have, you are actually reaching the level of an expertise. it is normal to face issues like this while just starting. You must continue until you get good result.

    Now, looking at your question, there are many reasons why you might have inconsistent result. If you are working with cells, your troubleshooting starts from the cell culturing technique. The question is were your cells in perfect condition before harvest? Are you sure you are not using infected cells. Hmn! The reason is that infected cells would also give you bands if you are good at WB but it might not be consistent, what I mean is that it might not give the expected blot trend.

    If you are sure of that, then you need to troubleshoot your extraction procedures. This step is also very important as you know that cells are very fragile. Are you sure you do not damage the cells during trypsinization and harvest? Are you sure your steps of protein extraction are perfect?

    Looking at your blot, it may be that the loading of the protein was not proper while running SDS page. For the control, I suggest you dilute the antibody the more. it seems the antibody is just too high. The same challenge might be applicable to your desired protein but if that is the issue, are you sure you loaded the appropriate volume of protein per well

    You can use the formula n=cv; where n = the number of moles of the sampl/well, c= concentration you got from your calculation curve after you've multiplied with both  the loading buffer constant and your dillution factor and v is the volume of the samples you wanna load. Therefore v = n/c. I always load 60microgram/microlitre. So, you can divide the 60mg/microL by the concentration of the sample to know the volume you will load per well. You need to load the same volume per well. You may go for the lowest volume after your calculation because all the protein wont give you the same volume but it may be very close.

  • Temitope Adelusi added an answer:
    I am getting dark blobs (bands) on my western blot membrane. Does anyone know what this means?

    I perform western blot according to LiCOR odyssey protocol. When I scan my membrane on odyssey CLx, I get dark blobs or bands at around 65 KDa. It seems to show up when I use some antibodies but with other antibodies on the same samples, I don't get these dark bands. Has anyone faced the same issue? Any insights would help.

    Temitope Adelusi

    The major challenge with this type of issue is the dilution of the primary antibody. I think, with your explanation different antibody might have different dilution factor. You may have to pay attention to your dilution factor. Some antibodies may have 1microlitre:500microlitre while some may have 1:1000 and if you are using a control like beta-actin, I always use 1:2000 and it gives clean beautiful bands.

  • Temitope Adelusi added an answer:
    Any advice on an SDS-page problem?


    I have trouble with running gel of western blot. I ran my protein with 12% polyacrylamide gel, 30mA for 1h. After I ran, I found bands expanded (see the picture). Can you tell my what happen and what should I do to solve this problem? Thank you very much. 

    Temitope Adelusi

    For SDS page, I always use 30 minutes, 60V for the proteins to leave the stacking gel but, most times I realize my proteins always leave the stacking gel even before the completion of the time. To resolve the proteins in the resolving gel, I use 100V for 2 hours. I always stop the process manually when I realize the markers are clear enough for me to cut out my protein of interest lane and i always use a resolving gel percentage that allows clear seperation between the markers. This is critical for clearity.

    With the picture I am seeing, I think the volume of protein loaded might be too much. I think. This picture makes me remember the first time I ran SDS page, that was the exact issue that occured. And you must note that you need to load the same volume of protein in each well. I always load 60microgram. Some of my colleagues do load 50microgram. I think you need to do your calculations properly. I think!

  • Tulika Tyagi added an answer:
    Does anyone have experience with multiple b-actin bands in western?
    I am using b-actin (monoclonal antibody from Sigma #A1978-200UL) as a loading control in my western but I am getting multiple bands (highlighted in red boxes in the file attached) instead of one. I use Typhoon imager to visualize the bands so it can't be because of movement of the film.
    Tulika Tyagi

    I was using BME but switched to DTT and it works good for me

  • Rayna Gasik added an answer:
    Any suggestions on why FGFR degradation experiment in HEK cells is not working?

    Hi, I am studying the effect of different mutations of a protein on FGFR degradation. Here is the layout of my experiment:

    HEK cells were cultured to appropriate confluency and then plated in wells. The following morning they were transfected with DNA (FGFR combined with either empty vector, or my mutations). The next day, media was removed and cells were serum starved for 2 hours, treated with 20 μg/ml cycloheximide for 1 hour and then stimulated with 100 ng/mL FGF for time periods of 0, .25, 1 and 3 hours, after which they were lysed. Samples were analyzed using SDS PAGE-western blotting. Blots were incubated with primary anti-v5 and anti-actin antibodies and secondary anti-mouse 680 antibody. 

    I was originally using half the concentrations of FGF and cyclohexamide, and trials were showing increase in FGFR, not degradation, which doesn't make sense. I tried doubling the concentration of each, and it worked...for two trials. The next trials showed increases in FGFR again in the first 15 minutes with eventual degradation after 3 hours. If anyone has suggestions or ideas about what might be happening, it would be much appreciated. I started with a fresh batch of cyclohexamide, so it should be working, and I shouldn't see increasing protein levels. 



    Rayna Gasik

    Hi, thanks for your response. I did try increasing the cyclohexamide, and used the same concentration. I have not made it fresh each time though, or checked the degradation of another protein. 

  • Jingjing Jiang added an answer:
    Why don't I see a band of my FLAG tagged protein in western blots?
    I have constructed two versions of my GOI with and without a FLAG tag and cloned them into the same vector. However, when I transfect HEK293 cells with my two different constructs, I can detect my protein in both cases with an antibody against my protein itself, but I can't get a FLAG tag signal with an anti-FLAG antibody. I even see a little shift of the band of my FLAG tagged protein.
    What encourages me is that the FLAG tag is expressed along with the protein. I've previously constructed FLAG tagged proteins in non-mammalian systems and they always worked perfectly fine. The DNA sequences of my constructs are confirmed.
    I'm using the anti-FLAG AB from Cell Signaling (1:1000 overnight), which detects the same epitope as Sigma's AB. I do block with 5% milk in PBST. My protein is approx. 15kDa.
    Thanks for your help!
    Jingjing Jiang

    Hi Rafael Garcia Tavares,

    I tried but I didn't get the result as expected. Later I realized it was a problem with the vector promoter strength. Because when I transfected the flag-tagged vector in a different cell line (HepG2), instead of 3T3L1 cells. I can detect flag without difficulty. It is not a problem with transfection efficiency since I used electroporation,which resulted in 80% or higher efficiency. So in my case, it is different from the question which Jose and you have faced.

    I think Jose's method is still worth of trying if you are facing the same problem.

    Good luck and sorry for the delayed reply.


  • Andreia N Carvalho added an answer:
    Can anyone recommend a good source for purchasing NrF2 antibody (for western-blot analysis) ?
    I have some problem for Nrf2 detection with total proteins and nuclear proteins extracts
    Andreia N Carvalho

    Hi Kritika

    For immunocytochemistry I used the Nrf2 antibody from abcam because it's the one that worked better.

    I can't remember if the one from R&D also worked for immunocytochemistry...

  • Carlos B. Rueda added an answer:
    Does anyone know any protein to be used as loading control of endothelium in western blot?
    I have been using b-actin as total loading control of protein but since loosing the endothelium layer is quite easy, I would like to find a protein expressed only in the endothelium.
    Carlos B. Rueda

    Thanks Cristina!

    I will see what I can do



  • Victoria E. Palau added an answer:
    Why are there double bands on western blots for mtor and p-mtor ser2448? Is there a biological explanation?

    I am unsure of the cause. It is reproducible across multiple blots and cells lines with both the phospho-specific antibody and the total mTOR antibody. Thanks for the help!

    Victoria E. Palau

    You may want to add a phosphatase inhibitor to your lysis buffer as well. It depends on the inhibitors you are using, but if you are looking for total protein, the recommended amount of protease inhibitors plus half of the recommended phosphatase inhibitor cocktail usually gives you clean bands.

    I use the Cell signaling antibody for mTor on several human cancer cells and have not seen these double bands.  

  • Aftab Alam added an answer:
    Can someone provide an Immunoprecipitation protocol for endogeneous Drosha and its partners and further western blotting as well ?


    I am looking for a working protocol for Endogeneous Drosha Immunoprecipitation and it´s Western blotting.

    I have magnetic beads  (dyna beads) as well as anti-FLAg M2 magnetic beads.

    I would be grateful if you could share the protocol with me.

    Thanks a lot

    Aftab Alam

    Generally for IP we do not do Ponceau, it can be a reason. But in input you will get band irrespective of ponceau. Silver staining is again tricky for mass spec analysis. If you want to do mass spec do 3-4 IP and pool them and run on the gel. Stain with coomassei, cut the band and go fro mass spec. 

  • Russell Linscott added an answer:
    Is there an alternative company for 4HNE antibody?

    I have been carrying out western blots using anti-HNE from alpha diagnostics, sometimes, I get bands and at other times my membrane is empty. Has anyone used this antibody and has any suggestions or recommendations to make?

    Russell Linscott

    If you need more sources than mentioned by Jan, we can provide several. Just search here http://www.linscottsdirectory.com/search/antibodies for 4HNE and follow the "More Info" links to the suppliers' data pages.

    Good luck!

  • Tanmoyita Nayak added an answer:
    Can someone help me with a low MW protein blotting problem?

    Hi all,

    I am working with an intracellular domain of a type1 transmembrane protein. This intracellular domain is really small (9 Kd) and contains a predicted NLS.  I made two constructs (with two different expression vector) of this ICD with and without its NLS sequence (I just deleted the first 9 amino acid of the ICD) to understand the functionality of that NLS.

    I performed western blot to check the expression of my constructs.

    Every time I am getting nice band from the ICD from both vector but not from the ICD w/o NLS constructs.

    I generally run 4-12% bis-gradient gel and blot for 2 hours at 200mA with whole cell lysate.

    Is it something to do with my western blot protocol or my ICD w/o NLS is not really stable?

    p.s: I have done ICC also and there I got nice stained cells from this ICD w/o NLS constructs and apparently the staining spared the nucleus.

    If anybody has any expertise with low MW protein blotting or with NLS constructs, please share your experience.


    Tanmoyita Nayak

    Thank you Xavier! I will try it next time with boiling PBS!

  • Juan Carlos Jiménez added an answer:
    Did anyone use the Western Blast Chromogenic blotting amplification system ?

    Did anyone used the Western Blast Chromogenic blotting amplification system ?

    I have to use it to amplify the signal of HRPO of my secondary antibody but i still have troubles with the protocol. 

    Is there anyone having a nice protocol of this technique. 

    I'm using the a Kit from Perkin Elmer  NEL761001KT


    Juan Carlos Jiménez

    I tested this system , you can use  the antibody biotinilated 1:1000 and after  avidin-HRP  1:2000  for 1 hr   that work  

  • Andrea Dimet added an answer:
    What experience do you have with the WES system from Protein Simple?

    I am interested to know how it compares to standard SDS-PAGE/Western Blotting procedures in terms of sensitivity  (using grey x-ray films), separation quality, handling and overall costs. Any critical issues?

    Andrea Dimet

    The cost is similar, though you are made more aware of it with the Simple Wes because of the upfront cost of the kit. You can dilute antibodies quite a bit more with the Wes than SDS-PAGE, so you save a good deal of money there. One disadvantage of the Wes is that samples are run in individual capillaries, thus there can be slight variability between capillaries-- unlike on an SDS gel where all samples are run within the same gel and transferred to the same membrane. Another disadvantage is that the kits they sell only have mouse and rabbit secondaries, so a secondary from any other organism will take some time to troubleshoot. Handling isn't overly complicated, and the Simple Wes definitely saves you time.

  • Arjen M Krikken added an answer:
    Does anyone have a protocol for Western blot yeast proteins without the use of glass beads?
    We work with a two-hybrid system for studying the interaction of viral proteins and use for this purpose, the yeast strain Y 153. We have our antibodies to viral proteins, and want to detect their fusion with the DBD and AD domains Gal on Western blots. But we don’t have glass beads. Who knows the alternative methods for lysis of yeast cells?
    Arjen M Krikken

    Preparing crude extract of yeast cells in presence of TCA

    To be used for Hansenula polymorpha, Pichia pastoris and Saccharomyces cerevisiae

    Published in:   Baerends, R.J.S., Faber, K.N., Kram, A.M., Kiel, J.A.K.W., van der Klei, I.J., and Veenhuis, M. (2000) J. Biol. Chem. 275, 9986-9995

    Goal:             Preparation of protein sample for SDS-PAGE analysis. Proteins are immediately denatured by TCA which prevents enzymatic modification of proteins (including proteolytic degradation, dephosphorylation, deubiquitination, deglycosylation).

    Materials:       1.  Eppendorf centrifuge

                         2.  -80 oC freezer

                         3.  Spectrophotometer

    Solutions:       1.  demineralized water

                         2.  12.5 % (w/v) Tri chloro acetic acid

                         3.  cold 80 % (v/v) acetone (cooled at - 20 oC)

                         4.  1% (w/v) SDS in 0.1 M NaOH

                         5.  4x SDS sample buffer

    Method:          1.  Cultivate yeast cells and harvest 3 OD660 units a), spin-down the cells in 2 ml Eppendorf tubes 1 min 14000 rpm, room temp.

                         2. Wash cells once in 900 μl water. Vortex cells, centrifuge again as above and remove supernatant.

                         3.  Resuspend the cells in in 400 μl 12,5 % TCA.

                         3.  Freeze samples at -80 oC for at least 30 min b).

                         4.  Spin down the cells at rT, 14000 rpm (Eppendorf microcentrifute), 5 min.

                         5.  Wash cell pellet twice with 500 ml cold 80 % acetone c), spin for 5 min., 14000 rpm,                 rT.

                         6.  Remove carefully the supernatant and dry the pellets.

                         7.  Dissolve the pellet in 75 mL 1% SDS / 0.1 M NaOH d).

                         8.  Add 25 mL 4x SDS sample buffer and boil the samples 5 min. at 100 oC.

    Notes:            a)   3.0 OD units yield approx. 300 mg protein.

                         b)   The samples can be stored at -80 oC for usage later.

                         c)   1st time resuspend pellet with pipet and 2nd time mix with vortex.

                         d)      At this stage the cell walls dissolve due to the NaOH.

  • James Firth added an answer:
    How can I minimize the air bubbles during a western blot wet transfer?

    Hello everyone,

    I am right now facing the issue of air bubbles during the western blot wet transfer method. It is so frustrating that some silly air bubbles mess up the whole results. This is what I do:

    I run an 8% SDS-PAGE gel, use bolt & mahoney transfer buffer, use nitrocellulose membrane for protein transfer. The transfer conditions are 25V, 300mA, constant voltage at 4C, with constant stirring. I leave this transfer overnight. At the end of the transfer time, I remove the membrane and wash it with 50ml of distilled water 2x5min. Later, I stain it in Ponceau solution for 30seconds, followed by 2x5min wash with 50ml of distilled water. I take the utmost care to prevent any air bubbles while setting up the sandwich. I also use the teflon roller to roll out any air bubbles which may have been trapped in between the gel and the membrane.

    I am so at the dead-end of what other tricks I could employ to minimize this issue. It would be very helpful if anyone can come up with any suggestions (something which I am not already doing) to eliminate the risk of air bubble formation. Thank you! 

    James Firth

    Interesting, I just ran my entire 1hour transfer at 20V in a 4degrees cold room. Maybe I'll try cooling down the gel for 10mins before constructing the sandwich. How long do your transfers last. Thanks again

  • Wangsa Tirta Ismaya added an answer:
    How do I solve my western blot problem?

    Hi everyone,

    Can you suggest me, I have a problem in western blot, I got a single band lower than expected. The band was very clear and dark. Can I use the antibody in ChIP seq.......

    Wangsa Tirta Ismaya

    the signal should be the same as for electrophoresis. are you sure that your gels are homogeneous? I normally run two sets of samples in one gel, then split the gel into two: one for staining and the other for WB. the two should correlate.

    are you using monoclonal or polyclonal. perhaps what was detected is other protein that cross-reacted with your antibody (while your primary target doesn't work).

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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