Western Blot

Western Blot

  • Amy Schutz Geschwender added an answer:
    How do you choose a good antibody for western blot?
    Usually there are several (even more than 10 or 20) antibodies for a protein in the market but how do you know which antibody is better or best for your experiment? Just try it one by one? Sometimes the company shows a good picture about the antibody at their website, but the antibody does not work......Does anybody have a good idea?
    Amy Schutz Geschwender · LI-COR Biosciences

    It's really surprising how much your blocking buffer will affect Ab performance. You can go from no bands (or lots of spurious bands) to a beautiful blot by changing the blocking buffer. It may be worth a quick optimization experiment to try a few blockers and see if a different blocker can rescue your "bad" Ab. I've attached a link to a paper on this topic.

  • Ángel Astroza added an answer:
    Are phosphorylated proteins of insulin signalling pathway very labile?

    I have run western blots and have got clean bands for AKT, phospho Akts and others but I am not able to get clear bands on repeating the western from the same samples around 3 weeks later.

    My sample was C2C12 cell lysate.

    Ángel Astroza · San Sebastian University

    ...and dont forget  "using phosphatase inhibitors in the lysis buffer: combination of phosphatase inhibitors, NAF, Na orthovanadate, Na deoxycholate, Na pyrophosphate along with protease inhibitors (RIPA buffer with all inhibitors)....this is VERY IMPORTANT...!!!

  • Can someone help with availability of p53 antibody for western blotting providing dealers in India ?

    can someone help with availability of p53 antibody for western blotting providing dealers in India ?

    Kunnathur Murugesan Sakthivel · Karunya University

    thank you all

  • Kw Agashe added an answer:
    Level of Phospho gsk3b in western blot?

    I am trying to study the level of phosphorylation in GSK3b in my cell lysates but I cant find the positive control for the PGSK3b anywhere commercially available online. can you suggest what i can use as positive control in western blots

    I do want a quantitiative measure as James said above...and hence I am rethinking using LiCl or IGF1 as both would just give me a positive result for ser9 phosphorylation but wouldnt give me a estimate of the amount phosphorylated..like the best way would be to have a cell lysate say 1mg/ml which i know is completely ser9 phosphorylated ( got it as positive sample with kit)

  • Giulio Agnetti added an answer:
    How can I estimate the molecular weight if the equation of curve is not linear ?

    I run the marker and unknown protein, and i got the very clear band of the marker. The molecular weight of the marker are 10 - 250 kda. After making a graph for rf vs log MW, the equation is not linear, the r2 only 0,91. Could i use this equation to estimate the unknown protein? or , i have to use the other equation? Thanks for your kind attention and suggestion.

    Giulio Agnetti · Johns Hopkins University, University of Bologna

    I concur with Alice, with in-gel digestion and mass spectrometry (MS) you could have the exact molecular weight. Your main band is low enough that you could probably skip digestion if someone at your institution does top-down MS. Good luck and happy new year

  • David Ronald Cavanagh added an answer:
    Why do the stacking gel wells rupture upon sample loading during PAGE ?

    Hi guys,

    Hope everything is going well with you. So I am encountering an issue, in which select (usually one or two) stacking gel wells rupture following loading the samples (usually containing 10 micro gms total protein), and the wells start leaking the samples. I am using 12% gels by the way. Would increasing the volume of TEMED, and APS, while preparing the stacking gel solve this issue. Also it is worth noting that the sample leakage doesn't always happen right away, sometimes it occurs minutes following the loading. Many thanks

    David Ronald Cavanagh · The University of Edinburgh

    One or two things come to mind here. 

    1. Your wells have un-polymerised acrylamide in them - pleases make sure you have flushed the wells with buffer before use. Stacking gels are usually 3-5% acrylamide. 

    2. There has been some mechanical damage to the well walls during gel preparation. this can be checked by adding diluted 1:10 sample buffer alone to wells to observe leakage. 

    3. You are using too much TEMED and APS, thus shrinking the gel around the wells during polymerisation. This might cause the gel to shrink from the plates, causing leakage. 

    Hope this helps

  • Ricardo Espinola added an answer:
    What effects does lyophilization have on serum samples? Will lyophilized samples run better on a WB?

    I've tried an ELISA with mixed results so I would like to confirm the results by western blot.  However, serum does not run well on PAGE gels.  Any idea if lyophilization and resuspension in water will help?  I ran serum on a 10% gel and it was close to the worst gel I've ever run in my life.  If anyone has any helpful suggestions, I would appreciate them.

    Ricardo Espinola · U.S. Food and Drug Administration

    Hi Stephanie,

    would you mind sharing with us what is your research question? Are you trying to purify a protein from serum?

    Your clarification will be very useful to guide you about your concerns.

    Thank you,

  • Gaurav Kumar added an answer:
    Where is the main location of p53 within the cell?

    In the cytosol or nucleus? Which buffer is suitable to lyse the cell to detect p53 by western blot? Please help me.

    Gaurav Kumar · Kurukshetra University

    As this is a protein, it should be in cytosol. It is present in inactive form maintained by mdm 2 protein.

  • Afshin Samali added an answer:
    Has anyone used a working antibody for phospho-IREalpha?

    I'm looking for a good antibody for phospho-IREalpha. Novus and Abcam sell the same antibody but do not want to buy it unless I know it works. I plan to use it on rat tissue.

    Has anyone used it yet ? 

    thanks guys

    Afshin Samali · National University of Ireland, Galway

    We have tried these and other antibodies to detect phospho-IRE1alpha.  Unfortunately, none of the antibodies seem to work as one would expect.  Some groups use anti-IRE1 antibodies and look at a shift in mobility of the protein as an indication of phospharylation of IRE1.

    If your ultimate goal is to study IRE1's RNase activity, I suggest you to look at XBP1 splicing either by RT-PCR or Western blotting.  There are several good antibodies to detect spliced XBP1 in several species. 

  • Andreas Kist added an answer:
    When eluting from Magnetic Protein A/G beads with SDS sample buffer, do I have to use DTT?
    I'd like to investigate if my protein of interest is covalently bound to the protein which was caught by the antibody. Can I leave the elution buffer (which is my SDS sample buffer) without DTT? Or has DTT some effect on the elution?
    Andreas Kist · Max Planck Institute of Neurobiology

    At the end, it worked (kind of) without reducing agents, SDS and LDS worked well. In my case it elution efficiancy was dependend on experimenter's harshness; always resuspend beads as good as possible and elute with harsh shaking. Be kind of rude!

  • Sara Hassan added an answer:
    How to go about a Phospho-AMPK blot?
    I am trying to do a blot for phospho-AMPK on primary fibroblast lysates. My standard protocol says to block with milk, then put primary at 1/1000 in 5% BSA in TTBS, then secondary at 1/1000 in 5% milk. This has worked intermittently, but the bands are always weak. It has been suggested to me that milk may interfere with detecting phosphor proteins hence I should substitute BSA for milk in the above protocol. This gives me a high background signal, I can see the outline of the correct bands against the high background, so I am guessing it has worked to some degree. What should I do next - should I block in milk now and then proceed with the standard protocol or just put it back in the primary (in BSA) and then hybridize with my secondary made up in milk or is there another non-milk way of decreasing background?
    Sara Hassan · Dana-Farber Cancer Institute

    Hello guys, I have the same problem. I used Cell signaling for both AMPK and pAMPK. I added phospho-stop tablet and Halt protease inhib to my Pierce lysis buffer. I can only see AMPK bands with regular ECL. When I used super ECL, I saw faint bands at the same size for p-AMPK together with many non-specific bands with the same intensity. I am not even sure if this is the real band! I had some problems before with detecting p-AKT! I don't know what am I doing wrong?! Please help

  • Adham Sabri added an answer:
    Dna vaccine against?

    Hello

    I am trying to clone the G gene from Viral hemorrhagic septicemia virus.

    For this purpose I cloned the G gene into pcDNA vector.

    After that I transfected it into EPC cell line and check the success of transfection by fluorescence.

    For confirming the transcription I did a RT-PCR and for confirming the translation I did western blot.

    But I couldn't get any band from RT-PCR or from western blot.

    Can someone help me to solve this problem?

    Thanks.

    Adham Sabri · Pukyong National University

    thank you so much for your suggestion. i will try to follow them and i will tell your the results.

  • Lawrence Broxmeyer, MD added an answer:
    Which region in the brain is most affected in Parkinson's disease?
    I have a some frozen brain samples from PD patients and control. These regions are Frontal, Temporal, Parietal, Occipital and Cerebellum. Can anyone tell me which region of these would be more suitable to study the difference between the PD and control samples. Mainly I will be carrying out ELISA, western blot.
    Lawrence Broxmeyer, MD · NY Insitute of Med Research

    More areas of the brain than just the Substantia Nigra pars compacta of the Basal Ganglia are affected'/involved with Parkinson's disease. Key gray matter structures: the thalamus, the globus pallidus, and the subthalamic nucleus also are among those involved. In the brain, pathways and structural insults are interrelated.

    Alpha-synuclein aggregated in several parts of the brain in PD, but especially in the substantia nigra. As mentioned above, it all depends on the stage of the disease in that patient under study. Braak et al 2003 gives you a good idea of the clinical stage involved.

  • Tali Feinberg added an answer:
    Can anyone recommend a good automated western blotting system/device?

    I am considering the Amersham WB system and the BIO-RAD V3 Western Workflow™ (though not fully automated). Are there any others and can anyone share their experience?

    Tali Feinberg · Tel Aviv University

    I would recommend on Bio-Rad V3- it is simple and very reliabel and repreducible.

  • Jaime Arellanes-Robledo added an answer:
    What is the positive control for CYP2E1?

    I would like to analyze the expression of CYP2E1 through western blot. However I could not able to get the positive control mentioned in the antibody datasheet (Human fetal liver extract.). Is there any other commercially available positive control for CYP2E1?

    I would also like to know the housekeeping protein for microsomal fraction. Please help me out of this.

    Thanks in advance for the answers

    Jaime Arellanes-Robledo · Instituto Nacional de Medicina Genómica

    Yes, liver is a good one but if you can extract liver mitochondrial protein is going to be even better

  • Corrine Tan added an answer:
    Platelet isolation and activation - can anyone help?
    Currently, I am trying to detect expression of specific proteins in platelet samples. My protocol begins with blood centrifugation to get the platelet-rich plasma (PRP). This PRP is then split into 5 parts before another round of centrifugation so as to get the platelet pellet and the plasma: (i) 1 portion is for non-treated platelet, that I immediately lyse after PRP centrifugation (ii) 2 portions, that I resuspend in Hepes buffer after PRP centrifugation, are for treatment with (a) calcium ionophore (activating the platelet) and (b) HBSS (salt solution, serve as control), (iii) finally, the other 2 portions, that I resuspend in Hepes buffer after PRP centrifugation and wash two times with the same Hepes buffer prior to the same treatment with calcium ionophore and HBSS. After treatment with either calcium ionophore or HBSS, I spin again to collect the supernatant fraction that supposedly contains the platelet releasate. I also collect the platelet pellet for lysis. Eventually, I run SDS-PAGE for plasma, all platelet pellets and supernatant fractions together and probe for my protein of interest.
    I encounter some problems:
    1. On my blot, I saw prominent white negative intensity bands between 50 and 75kDa mark. And the size of my protein of interest is 63kDa, which is between 50 and 75kDa mark. Could these white bands actually be albumin? Or other residual plasma proteins?
    2. Even though, I could not detect the correct 63kDa protein band (which could have been masked by these white bands), I detected prominent bands at around 50kDa mark instead, just below the white negative intensity bands. Strangely, these bands were only present in the plasma and the non-treated platelet pellet (part i) as well as in the supernatant of non-washed platelet (part ii, the platelets were not washed 2 times before CaI or HBSS treatment). This observation makes me wonder if the detected proteins are actually coming from the residual plasma protein again? Because there were no protein bands for the platelets that were washed 2 times prior to treatment.
    I really hope somebody, with experience working with platelets, could give me some advice or feedback regarding this matter.
    Corrine Tan · National University of Singapore

    Hi Paula,

    You mentioned in one of the post that blood should not be collected in glass tube. Why is it so? The ACD collection tube from BD come in glass format only. 

    Thanks in advance.

    Cheers,

    Corrine

  • Sandeep Rajput added an answer:
    Why am I not able to detect cleaved caspase-3 by Western blotting?

    Hi!

    I am trying to detect active caspase-3 by western blotting in human cancer cells treated with etoposide. Although I am able to detect procaspase-3 and actin as a loading control, I cannot detect a cleaved form of caspase-3. The concentrations of etoposide are 25 and 50 microM, and the cells clearly undergo apoptosis (measured by PI staining). It seems to me that this is a technical issue.

    Does anyone have any tips how to detect active caspase-3 by WB?

    Briefly:

    Caspase was separated on 15% polyacrylamide gel (SDS-PAGE)

    35 or 45 micrograms of protein was loaded

    Proteins were transferred to PVDF membranes.

    Transfer buffer contains 20% methanol.

    Caspase-3 rabbit monoclonal antibody (8G10, Cell Signaling) 1:1000 was used

    Sandeep Rajput · Washington University in St. Louis

    You can try Cleaved PARP which is more broader apoptotic marker than caspase 3. You can also try immunocytochemsitry for cleaved caspase 3 on cells seeded on coverslips.

  • Marcela Laukova added an answer:
    The most appropriate nuclear and cytosolic fraction housekeeping gene?
    Could anyone recommend the most appropriate housekeeper for nuclear and cytosolic fraction? I want to isolate both fractions from frozen rat brain tissues and am wondering what is the best and most commonly used housekeeper to quantify changes in another protein of interest using Western blot?
    Marcela Laukova · New York Medical College

    Hi Jonathan, yes, I already did it and published successfully, though it depends whether you are using frozen or fresh tissue. With frozen one do not expect 100% purity since the cells are already disturbed. I used snap frozen brain tissue and got nice purity though nuclear fraction was little bit contaminated by cytosolic one. Please, see attached paper with the method I used and let me know how it worked for you. Also there is a kit from Qiagen or other company for fractionation, but I have not tried it. It also suggest to use fresh tissue if possible. Good luck.

  • Zhen Chen added an answer:
    Cell lysate preparation for western blot of HeLa cells
    I have been trying to do a western blot for S6K phospho protein in HeLa cells, but unfortunately I do not get much total protein to start with. I grow the cells on a 6 well plate until they get ~80% confluent, lyse them with lysis buffer containing 4% SDS and also containing lots of protease and phosphatase inhibitors. Scrape the cells off the plate and leave it on ice for around 20mins before centrifuging at 12000rpm for 20mins at 4°C. Can anyone suggest something I'm doing wrong? I have tried the same with primary rat hepatocytes and they work.
    Zhen Chen · Chinese Academy of Sciences

    ”This S10 extract (40 to 50 A260U/ml) was stored in small aliquots at -70℃,(Translation of Poliovirus RNA in Vitro: Changes in Cleavage Pattern and Initiation Sites by Ribosomal Salt Wash,1979)“,so how can I get the  50 A260U/ml?

  • Steingrimur Stefansson added an answer:
    Did anybody do western blotting of globin?

    I need to do western blot of RBC globin but i can't find how to extract globin from the cells. I will use anti-AGE antibody. Did somebody do something like that? Thank everybody for answers!

    Steingrimur Stefansson · HeMemics Biotechnologies Inc

    Hi Taras, point taken.

    I was thinking that you were analyzing hemoglobin from diabetic patients.

    What I meant with Hb that  "it usually does not develop advanced glycation end products in vivo." is that the RBCs have a high turnover in the circulation (20-30 days) that precludes them from developing more advanced Amadori and Maillard reaction products that can be classified as AGE.

    You usually don't find vascular proteins with AGE products because vascular proteins are turned over rapidly by the liver.

    Extravascular proteins, like collagens and elastin, however, have a long turnover rate (~years), are more likely to develop AGE products.

    Question. Why do you want to want to remove the heme group from hemoglobin? The heme is not involved in the glycation process as far as I know.

  • Jan Piwowarski added an answer:
    What is a suitable protocol for transporter protein detection using western blotting?

    Can you mention steps for the separation and detection of transporter proteins like MATE & MDR1....etc

    Jan Piwowarski · Polish Academy of Sciences

    Dear Ali,
    Your question is a bit to general to press the RG members to help you. Suggest you to find you the colleague fluent in western's at your university. To plan it you must take into consideration protein size, hydrophobicity, and of course equipment available to you. The antibodies choice is another huge issue. Excellent advises like Adam's above will be very valuable. Anyway to avoid the beginners faults friendly tutor nearby is required. The list of my and my students mistakes you will find below:)
    https://www.researchgate.net/post/Can_you_show_the_picture_of_your_worst_Western_blot_and_explain_the_reasons_for_this_disaster_please
    Keep figers cross - your Jan

  • Ganesh Shelke added an answer:
    Why am I not seeing banding on exosome western blot for Alix, Flot1, TSG101?

    I have attempted a western blot for SKOV3 exosomes and there are no bands present in the exosome sample (if there is they are in the incorrect MW location) and the cell lysate sample has nonspecific binding of the antibody. I used 18ug of cell lysate protein and 33ug exosome protein in each lane of the gel (NuPAGE 12%). For the sample preparation I used LDS sample buffer, RIPA buffer and protease inhibitor; vortex briefly, on ice for 10 min, vortex briefly then incubated at 70 deg C for 10 min then on ice until I loaded the gel. Transferred to PVDF membrane and visualized the protein separation with Ponceau staining. Then I used 5% NDMF to block for an hour and added the antibody to incubate overnight at 4 deg. The following day I rinsed with 1x TBST 3 times for 5 min each then the secondary ab (HRP) was incubated for 1 hr. The wash step was repeated and ECL substrate was incubated for 5 min then imaged using chemiluminescence. I am attaching a picture of the Ponceau staining (MW marker, CL, exosome; repeated 4 times total) and the resulting western blot with the antibodies. Any help would be greatly appreciated!

    Thanks!

    Ganesh Shelke · University of Gothenburg

    Hi,

    give a pbs wash to your extracellular vesicles (EVs) pellet to remove contributing serum protein (albumin) and do protein estimation again...by doing this u will get more  EVs protein per unit of total protein. Good luck.

  • Begum Erdogan added an answer:
    Is there a PDGF antibody (for PDGF ligand) that recognizes all PDGF isoforms?

    I was wondering if there's an antibody that recognizes multiple PDGF isoforms, such as AA and BB. Thanks!

    Begum Erdogan · Vanderbilt University

    Thank you!

  • Kai F. Albring added an answer:
    How can I measure prenylations on an IP of my protein?

    I want to measure prenylations, specifically geranylation. I have a FLAG epitope on my protein and want to do an IP and then blot for geranylations. However, I cannot find any geranylation antibodies. -does anyone know of usable antibodies?

    Kai F. Albring · Friedrich-Schiller-University Jena

    Hi Lykke,

    if are using a vector with some viral promotor like CMV to overexpress your FLAG-tagged protein of interest, you will most probably overwhelm the cellular machinery involved geranylation. Some of the protein might still be modified but it could be that the majority is not.

    If possible, you should try to do an IP of endogenous protein. This approach should yield more reliable information than trying to analyse modifications on overexpressed proteins.

    Best,
    Kai

  • Kellie Bowen added an answer:
    Has anyone used Enzo's CB1 antibody ALX-210-314-R100? How successful is it, and what ratio is necessary to develop a clear western blot?

    Using this antibody in HEK cells and having trouble getting consistent results. Wondering if anyone else is having the same issue. 

    Kellie Bowen · University of Wisconsin, Madison

    Do a protein gradient and a antibody dilution series.

  • Savita Shah added an answer:
    How can I fix unknown lines in SDS-PAGE electrophoresis?

    Dear,

    I take a SDS-PAGE electrophoresis gel. It appears some lines which I don't know what they are. The line connect all wells of gel and they are more than two.

    What's it and How to fix my trouble?
    Please help me.

    Savita Shah · San Jose State University

    Did you wash your cells/plate (if the cells are attached) before you lysed the cells to isolate your protein. Since we use pretty high conc. of FBS (10%-  which is really  BSA) in the media, if you do not wash your plate with PBS, you will always carry over this protein in your cell extract..When you repeat this expt. , wash you cells/plate throughly with PBS and then proceed for protein extraction..You should see this band (almost)gone

  • Jan Piwowarski added an answer:
    Is anyone working with FABP4 protein for Western Blot?

    Hi,

    I have problem for Western Blot for FABP4. I 'm using primary from R&D Minnepalls, Cat#1443. I couldn't get good bands. Can anyone help me?

    Thanks,

    Jan Piwowarski · Polish Academy of Sciences

    You have two antibodies at this page:
    Mouse/Rat FABP4 Affinity Purified Polyclonal Ab, Goat IgG AF1443
    Mouse FABP4 Biotinylated Affinity Purified PAb, Goat IgG BAF1443
    Which you are using?

    Anyway a goat is a nice source for the producers, since gives relatively large volume of serum, but the antibodies are rather poor quality in my experience. Suggest you rabbit or mouse produced IgG. Anyway with your enigmatic description it will be hard to help you as Muralidhar mentioned. The full procedure is requred.
    Regards - Jan

  • Mansoor Azeem Siddiqui added an answer:
    Which is the concentration of protease inhibitors for the extraction of muscle proteins?

    I will make extraction of muscle proteins for electrophoresis study. Does anyone have a suggestion for the concentration of protease inhibitors? Based on some studies, I'm thinking to use: benzamidine hydrochloride hydrate (1 mM), phenylmethanesulfonyl fluoride (PMSF) (1 mM) and EDTA (10 mM). Thank you very much

    Mansoor Azeem Siddiqui · International Centre for Genetic Engineering and Biotechnology

    It is better if you could incorporate Leupeptin , Pepstatin , Benzamidine , Antipain ,Chymostatin and also EDTA since muscles are calcium rich tissues.PMSF however is more useful if you need to block phosphatase activity.

    above mentioned formulations are time consuming to make so Its better if you use Thermo Protease inhibitor cocktail Tablet or liquid.

    Moreover the amount of protease inhibitors needed depends upon the amount of proteases in a given tissue and not as such the proteins.

    Hope it helps.

    Regards

  • Calvin S Leung added an answer:
    For Co-IP experiments, is it more ideal to have more bait or prey protein?

    I have been trying to do a Co-IP between Protein X and Protein Y. I tried pulling down for X, which is highly expressed in the cell, and probing for Y, which is not expressed as much, and I am getting a very weak signal in my western blot. Would it make a difference if I did the reverse IP?

    Calvin S Leung · University of California, Los Angeles

    Thank you for all the helpful replies! I am doing the reverse IP and will know the results soon.

  • Mohammad mohsen Mohammadi added an answer:
    How can we perform migration of peptide correctly in SDS-PAGE?

    Dear All,

    I am trying to detect synthesised peptides with SDS-PAGE. The peptides migrate with a big difference (larger) in SDS-PAGE gel in comparison with their real molecular weight. And they refused to migrate in a single band, which makes things even worse. Do any of you have suggestions for improving the detection, please?

    Thank you.

    Dear Lauisa,

    My peptides are ranged from 2 to 4kd. There is no Cyc in the sequence and I used DTT heating step. I do not understand what you mean to deprotect peptides.

    They migrate to 6 to 20 kd with normal SDS-PAGE. I am thinking about to try Tricine-SDS PAGE. And may also lower SDS concentration in Running buffer, run in a lower voltage.

    It will be helpful if you share your expertise.

    Thank you.

    ziguo

    Mohammad mohsen Mohammadi · Shiraz University

    hi. may be dilution of your sample is helpful.

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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