Western Blot

Western Blot

  • Ariel Orbach added an answer:
    1
    Phosphorylated ABCA1 antibody?

    Hi all,

    I am currently searching for an antibody against phosphorylated ABCA1 for western blot. I have a Novus anti-ABCA1 antibody (NB400-105) that is working quite well, but cant seem to find a good one for the phosphorylated protein.

    I found a couple online (http://www.emdmillipore.com/US/en/product/Anti-phospho-ABCA1-%28Ser2054%29%2C-clone-EPR2485%2C-Rabbit-Monoclonal-Antibody,MM_NF-MABS734 and http://biossusa.com/store/bs-12956r-hrp.html), but cant find any papers that used them, so Im not sure if they're any good. And all papers I've seen that looked at phospho-ABCA1 used either 32-P or ABCA1 IP and then phospho-serine to determine levels of phospho-ABCA1. We cant use radioactive labeling in our lab, so I may try the IP protocol, but would prefer to just find an antibody against p-ABCA1.

    If anyone knows of a good antibody for this, or a paper that used one, please let me know!

    Thank you so much for your help!

    Ariel Orbach

    Hello,

    Do you know the phosphorylation site?

    Abcam has an antibody that may work for you.

    Anti-ABCA1 (phospho S2054) antibody [EPR2485] (ab125064)

    Good luck

  • Radosveta Koldamova asked a question:
    New
    Does anybody have an experience with GE Healthcare ImageQuant LAS 500 for Western blotting?



  • Steingrimur Stefansson added an answer:
    2
    Is it possible to get signal for Actin on a western blot prior to incubating with a-actin antibody?

    I have a western blot which I incubated with an antibody anti-ADH 7. 

    I had a faint band appearing at approx 42 kDa. When I incubated with anti-actin afterwards, a band at approx 42 kDa appeared, with the same size and same shape of the band that had appeared prior to incubating with the actin antibody.

    Could my antibody be cross-reacting to actin? Could this just a coincidence and they are just different bands with the same weight and same size?

    Steingrimur Stefansson

    Hi Diogo, could you provide more detail.

    Was this 42K band visible from Ponceau staining of the blot prior to ab staining?

    As Adam said, the antibody manufacturer might be bad.

    You could also try to run native PAGE of your sample and blot. The ADH dimer should show up.

  • Işın Tuna Sakallıoğlu added an answer:
    3
    What concentration are you using for primary antibody PARP on Western Blot while using MCF-7 treated cells?

    In general for HCT116 cells we are using 1:2000 PARP to see parp cleavage. I have not done Western for parp cleavage with mcf7 before but i specifically know that it is cell-line dependent, and also depends on which antibody you are using. 

    Işın Tuna Sakallıoğlu

    Thank you I will consider these for my further protocols also for HCT116. I have seen PARP cleavage on MCF-7 treated cell on some of the papers its so strange to hear that, I will dig into that. 

  • Parthasarathy Chandrakesan added an answer:
    3
    Problem with phosphoprotein detection? BSA vs Milk and TBST vs PBS??

    We have been having problems with high background in our western blots for phospho-proteins even though we are using BSA. We found that certian lots of BSA give high background. What are your inputs? we use TBST.

    Parthasarathy Chandrakesan

    Either you can reduce the % concentration of your blocking agent and or you can switch using non-protein blocking agents. On my experience 3-5% BSA works better for phospho-protien detection.

  • Suman Kumari added an answer:
    6
    What may be the reason that I am getting gene expression but not protein expression?

    Hi,I did my qPCR experiment in kidney tissue for alat2 gene ( mitochondrial alanine aminotransferase) and I got 3-4 fold increase as compared to control, but when I am doing my Western Blotting ,I am not getting any protein bands at al.Please suggest any reason or solution for this.,thank you

    Suman Kumari

    Good Morning sir and Thank you so much  for your paper.

  • Geetha Samak added an answer:
    3
    How to extract protein from Perfused brain for western blot?

    Hi,

    I am going to perform  brain clearing CUBIC protocol and before that I need to do western blot with the perfused brain tissue. Does anyone suggest me the protocol for the lysis of brain tissue, which buffer has be used. I have done western but not the initial lysis step and that too for Brain tissue. I am very new to this and would appreciate any suggestions.

    Thanks in advance.

    Geetha Samak

    You can use any cell lysis buffer with protease and phosphatase inhibitors ( Lysis buffer D,CS, RIPA etc.,). It also depends on which protein you are looking for membrane/cytoskeletal/cytoplasmic ?

  • Valerie Orr added an answer:
    5
    NO/weak bands with western blotting...comments/suggestion?

    Hello everyone

    I did western blotting a couple of times and it always worked. After a month when I tried to repeat the exact same protocol; most of the times I didn't see any bands while other times it was just non-specific binding. I am looking for  the same protein of interest (17KDa) . As far as I remember, no changes have been made in the protocol or reagents. There is no problem in protein transfer. Also, I don's see band for positive control recombinant protein. I am doing semi-dry western blotting and using Bio-rad ECL clarity for detection.

    To troubleshoot the problem,

    1. I changed blocking reagent from 5% BSA to 5% milk.

    2. Prepared fresh primary antibody dilutions in 5% BSA (suggested by company).

    Could it be poor quality of BSA/ ECL?

    any comment/suggestion is appreciated. Thanks

    Valerie Orr

    When I would trouble shoot a western blot in the past I always tried doing a dot blot first with the following controls (basically activate the membrane, add a small (1uL) drop of samples and let it attach). 

    I would do dots of the positive control (your purified protein that should work), the primary antibody, and the secondary antibody. After you let the proteins attach, block the membrane as usual and do the incubations in the primary and secondary antibodies. Sometimes I do a bunch in parallel and use different concentrations and combinations to optimize the response. 

    If your secondary antibody is detected, your detection method is working.

    If you primary antibody is detected, your secondary antibody & detection is working.

    If your protein is detected, everything is working. 

    Hopefully you can see how you can trouble shoot quickly (and optimize your antibody concentrations) doing a dot blot with those controls since dot blots only take a few minutes to set up instead of doing SDS-PAGE and a transfer just to have it fail. 

    Afterwards if you are detecting your protein, and it still doesn't work on your western, then its your transfer. Try staining your blot to see if the proteins are there. 

  • Abhishek Verma added an answer:
    5
    Can we co-incubate the primary and secondary antibodies at the same time for Western Blotting ?

    Generally, we incubate the blocked membrane with the primary antibody, wash away the unbound primary antibody, then incubate  with the secondary antibody which binds to the primary antibody, and  detect  the protein of interest on the membrane. Since the primary antibody binds its epitope with its variable domain, whereas; the secondary antibody binds with its constant domain, there should be no reason that mixing these two antibodies together will interfere with the binding.  Can we incubate the blocked membrane with both primary and secondary antibodies at the same time for Western Blotting ? If it works, it will save time.

    Abhishek Verma

    I think this will work good if primary antibody is highly specific. Moreover you need proper TBST washing followed by incubation. Some antibodies are so good to give good signal after even one hour of incubation at room temperature. So I think this condition depends on specificity of antibody.

  • Mahitab Elsayed added an answer:
    3
    Dose anyone have a protocol for sampling preparation for treated cell culture for western blot?

    sampling preperation

    Mahitab Elsayed

    I want to know how can I prepare the cell culture dish in 6 well plate , because I am usually working on 96 well plate, and how can I add my reagents in it 

    what is the protocol for the 6 well plates and needed volume of cells and reagents per well

  • Michelle Smith Johnson added an answer:
    37
    Best way to resuspend Acetone-precipitated protein for Western Blot?
    I'm using QIAGEN kit to extract RNA and to better use these samples, I decided to extract protein as well, which QIAGEN recommends to do so by adding acetone to the flow through.

    Judging by the size of the pellet I've been able to collect great amounts of protein, however, when I try to resuspended in RIPA buffer or PBS alone, the pellet barely dissolves.

    Does anyone have a suggestion in how to resuspend the pellet or what buffer to use?
    Michelle Smith Johnson

    http://www.monzir-pal.net/Bioseparation/Lectures/L9.pdf

    The above link gives an excellent description of which buffer will work to re-solubilize proteins and how each method of protein precipitation works.  I can tell you from experience that RIPA buffer does not work that well on methanol precipitated proteins.

    Best of Luck.

    Michelle

  • Chao Huang added an answer:
    3
    Does anyone recommend any markers for mouse exosomes for western blot?

    Hello all, 

    I tried to isolate mouse plasma exosomes by using ExoQuick, after examined with a panel of antibodies(CD63, CD9, CD81 and HSP70), only the CD63 shown positive in western blot. I am wondering does anyone have tried to measure mouse plasma exosomes and recommend any antibody? Thank you so much.

    Chao Huang 

    Chao Huang

    Thanks for the response!!!

  • Patrick Baril added an answer:
    22
    Does anyone have experience using the Trans-Blot® Turbo™ Transfer System from Bio-rad?
    So I am considering using the trans-blot turbo system over the traditional tank system. Transfer of proteins using the tank system can be very efficient, but takes long and bubbles are always a problem. So anyone have experience using this trans-blot turbo system?
    Patrick Baril

    As for other colleagues, I must say that the transblot turbo system is working quite well, fast and efficient protein transfer method. The only problem is to transfer high molecular weight protein, larger than 80 kDa. We get used to stain or PVDF membrane right after the blotting and we can easily see that the efficiency is quite low compared to smaller proteins. We tested several settings and improved a bit the transfert efficiency but not enough to detect slightly expressed proteins. The price is also a problem but in term of fast, reproducibility and efficiency (for low molecular weight proteins again) is fair enough to have it.

  • Adam C Munhall added an answer:
    7
    Are Biorad's stain-free gels compatible with Merck Immobilon Low Fluorescence PVDF membranes?
    Has anyone used these two products together successfully? I was told I need to use a low fluorescent (LF) membrane when trying to visualize the proteins after transfer from a stain-free gel (since my normal Immobilon-P membrane gave lots of background fluorescence), but I don't want to use Biorad's LF membrane because it's very expensive and more than double the price of Merck's LF membrane. I'll be using the FastCast stain-free gels from Biorad. Any advice is much appreciated!
    Adam C Munhall

    Since the initial post of this thread I have purchased the Immobilon FL membrane and in my hands is much better than Amersham Protran 0.2 nitrocellulose with respect to autofluorescence background signal using a Licor Odyssey. Very impressive. I've also used Bio-Rad Criterion precast gels with this membrane successfully using the wet transfer method at 20V overnight (< 100mA).

  • Marcela Laukova added an answer:
    3
    Is there any reliable antibody against corticotropin-releasing hormone (CRH) for Western blot?

    Hi guys,

    does anybody know any CRH antibody which would work for western blots giving a nice and specific signal, especially in brain tissues (PVN, hippocampus)? Any advice is welcome. Thank you.

    Marcela Laukova

     Thank you a lot, Russel. Well I am aware that there are multiple anti-CRH antibodies but nobody would like to spend a tremendous time and resources to test each one. Additionally, we all know that companies selling them are not always honest once showing representative blots which are not convincing and the same with article resources, which do not even mention or use that particular one ;-)

    So, it would be great to get a reference from users who actually tried any of them themselves. 

  • Saleh Alkarim added an answer:
    2
    Can anyone suggest a good CSE/CTH antibody for Western Blot?

    I have used ABCAM and ThermoFisher and both do not work that well. Using it on Human samples, mainly trophoblast cells.

    Saleh Alkarim

    Hello

    i agree withe Syed A Ali 100% . look at links here and try with R&D system , abcam , biocompare , cellsignal antibodies companies .

    Good Luck

    + 5 more attachments

  • Zhaoxue Ma added an answer:
    36
    Protein doesn't migrate through the SDS PAGE. Can anyone tell me why?
    I did a Western Blot and I detected a flag-tag on my protein (see pic). The size of my protein is 56 kDa, which corresponds with the lower band. The upper band looks like it didn't run in properly. I loaded a lot of protein this time around.
    Any suggestions on how I can do a better job on this?
    Zhaoxue Ma

     Hey Christoph,

    I have the same problem with you. The protein aggregate on the top of the running gel. In your lammli buffer, you add both DTT and beta-mercapto? 

    Thank you.

  • Wassim Eid added an answer:
    3
    Can we use luciferase assay cell lysate for western blot? if anybody has used, please share protocol?

    I have performed luciferase assay and I want to do western blot using the lysates.

    Wassim Eid

    In theory, yes ...


    check this detailed information sheet from Promega.

    In practice, I would not recommend, because-as mentioned by Hamadi, there is a risk of digestion by proteases if u did not add the proper inhibitors.

    you can try to add the inhibitors when you lyse, but am not sure if this affects the readings of the luciferase assay or not.

  • Chethan Reddy added an answer:
    4
    Commercially available Arc antibody for western blots?

    Hello,

    Does anyone know of a good commercially available antibody for quantifying Arc (Arg3.1) expression from tissue samples?

    Thank you.

    Chethan Reddy

    Thanks for the input Claudia. I think there are two "Arc"s. What I was looking for is an activity regulated protein,Arc, also known as Arg3.1. The one you suggest is different.

  • Jeffrey Lakritz added an answer:
    7
    How to boil samples for Co-IP with magnetic beads for WB?

    Hi, 

    I am trying to perfom a Co-Immunoprecipiation and I am not sure about one of the steps in the protocol. 

    I incubate my lysates with the antibody againt my protein of interest over night and on the next day in Protein G magnetic dynabeads, then I wash them and add LDS buffer + BME. 

    Before letting my samples run in a Western Blot I have to achieve to things: 

    1. remove the magnetic dynabeads - the protocol says to boil the samples at 70°C for 10 min in order to do that 

    2. reduce the proteins in my sample, so that binding partners will separate - the protocol says to boil the samples at 95°C for 5 min in order to do that

    Can I combine these two steps of boiling? Meaning boil the samples for 10 min at 95°C? Or will this cause damage to the beads? Or could I boil the samples for 10 min at 70°C? Or is that temperature to low in order to reduce the protein with BME? 

    Thank you very much for your help! 

    Jeffrey Lakritz

    What questions do you want to answer? If you do not care what proteins are bound with the protein your Ab is bound too, then heating with SDS (or LDS) will be fine.  If the co-IP proteins are of interest, perhaps a less severe method of removal from beads would be useful.  We used pH 2.5-3.0 citrate or other buffers to remove proteins from beads.  We then treated with SDS loading buffer and ran proteins on gel.  Cut bands to determine who was who.  Then go back to use WB to demonstrate which proteins came down?  Depends on what you are looking for.  Do you know whether the proteins Co-IP are covalently linked?  If not, then only reason to reduce would be to demonstrate protein by MW and by WB.  Are you just using 1D or 2D?  Jeff

  • Mitali Das added an answer:
    8
    Integrin alpha 3 antibody for western blot
    I am having problems to find a good anti human-integrin alpha 3 antibody that works on western blot.
    We are using one from Santa Cruz (clone C-18), and I tested everything: dilute in BSA in TBS, Milk in TBS, different lysis buffers, different dilution of the antibody, different amount of protein loaded and I could not get any detectable band in my membrane.
    Mitali Das

    Hi Chiara,

       Use this antibody from BD Biosciences (Cat# 611140) to detect human beta3 integrin in a western blot (at 1:5000 dilution):

    http://www.bdbiosciences.com/us/reagents/research/antibodies-buffers/cell-biology-reagents/cell-biology-antibodies/purified-mouse-anti-human-cd61-1integrin-beta-3-chain/p/611140

       I just used it last week and worked very nicely.

     Hope this helps,

        Mitali.

  • Junjie Zhang added an answer:
    2
    NFATc2 (NFAT1) westernblot in Jurkat T cells?

    Hello Dear colleagues,

    I need some help with NFATc2 (NFAT1) westernblot in Jurkat T cells.

    I induced Jurkat T cell activation with CD3+CD28 antibody and goat-anti-moue IgG then collected the cells at different time points and checked NFATc2 with WB.

    I lysis the cells using my lysis buffer (1% triton X-100, 150 mM NaCl, 50 mM Hepes pH7.5, 10% glycerol with protease inhibitor), then use supernatant to do WB. 

    With same samples I can detect NFATc1 and another kinase very well. But the NFAT1 bands seems odd. I got 5 major bands 100 kD, 80 kD and 50 kD. But according to the datasheet of the company, the band should be around 110-130. I did get some weak bands at around 120 kD, but it seems not the major band.

    btw,  I am using the NFATc2 antibody from Santa Cruz 4G6-G5, which is widely used to detect NFATc2.

    Any suggestion will be appreciated.

    Junjie Zhang

    Hi Andre,

    I think the antibody I am using (4G6-G5) doesn't differentiate the phospho-and dephospho-NFAT1. 

    You think the band I got around 100 kD is the dephospho-NFAT1? I didn't use the phosphatase inhibitor, and I will include next time.

    Thanks for the pic.

  • Yunjiao Zhu added an answer:
    7
    WB Protein concentration (ug/ul) too low to fit 60 ug total protein in 14ul gel. How to fix?

    I work with postmortem tissue. When conducting a western, we use 60ug total protein. Meaning 14ul of 60ug total protein solution is added to 14ul loading buffer, creating 28ul that is used to fill two wells. 

    I am testing the protein concentration to determine how much of the supernatant needs to be added to water to result in a 60ug/14ul solution. My tissue samples have too low of a protein concentration (ug/ul) to be able to fit 60ug total protein into 14ul based on our current protocol. I would really appreciate some help! Thanks

    Yunjiao Zhu

    Using a more concentrated loading buffer is probably the easiest solution that you can try. I used to use a 5X SDS loading buffer and it worked well. See recipe here: http://cshprotocols.cshlp.org/content/2010/10/pdb.rec12340.full?text_only=true

  • Arthur Fischbach added an answer:
    4
    Can someone suggest a good and specific anti-dsDNA antibody?

    I am looking for an anti-dsDNA antibody. (Could also be an anti-ssDNA antibody). Can someone suggest a good and specific one? I would like to precipitate sheared Protein-chromatin complexes with it.

    Arthur Fischbach

    Thank you, Robert!

  • Tie Duerna added an answer:
    8
    Why human serum used as 1st antibody always cause high background in western blot?

    My western blot protocol was :

    • Sample:normal human epidermal extract, 25 ug/lane
    • 7.5%PAGE, semi-dry transfer to nitrocellulose membranes.
    • Blocking buffer:5% skim milk in TBST(0.5% TWEEN 20)
    • 1st antibody:Human serum 1:20 diluted in blocking buffer,4 degree ,O/N
    • 2nd  antibody:Rabbit  anti-human IgG(1.3 mg/ml )1:40000 diluted in blocking buffer,RT,1h.
    • Washing step: TBST(0.5% TWEEN 20),10 min,2 times;5min,1time.
    • ECL prime detection.CCD camera expose.4 sec.

    loading pattern:

    • lane a and lane b: human serum. 
    • lane c: without 1st antibody(human serum).

        I attach the result,as you can see ,the background from serum was dark,and My western blot was aimed at the high M.W. between 250 kDa to 100 kDa.

      I expected lane a had bands between 250kDa to 150kDa, lane b was set as normal control, and there was a strong nonspecific band .

      I have tried change blocking condition at 4 degree O/N;change blocking buffer (5%BSA);diluted 1st antibody(human serum) further ,while further dilution weaken the specific banding,and those can't help me to get expected result.

      Is there any advice about reduce the background caused from human serum?

    Thank you for your help!

    Tie Duerna

    Dear Duco

           My next plan was Affinity purification,using IP(pathogenicity IgG) or just  separate whole IgG.As your suggestion ,I will consider affinity-purify the lane b's serum.

           As for the rabbit serum blocking test:

           I have no ready-to-use normal rabbit serum ,but I have normal donkey serum ,and my secondary Abs have no cross-reacts with donkey serum by rocket immunoelectrophoresis, so I tried it .

             I applied 1% donkey serum for blocking ,the background were much clear than other blocking( BSA,Skim milk,PVP, some blocking product from company) when expose for 4sec.But the blot still not suitable for longer time expose that bring's nonspecific band from 2nd Abs.

        I guess there are something(such as amino acid,AA )in human serum and contain to the membranes,and 2nd Abs recognized them and bring the background.Using rabbit or duckey ' serum blocking those aperture in membranes ,instead of human serum's component to fulfill the aperture, this reducing the background.

     Thank you for you advice!

      Best regards!

      DUERNA

  • Zaynab Saad added an answer:
    2
    Is their any differences between ECL western blotting apparatus and gel band fluorescent detection apparatus?

    Hi ... in lab. I have band ge detectionl fluorescent apparatus and I'm undergoing to do western blotting using enhanced chemiluminescence (ECL) 

    My question is ... can i determine the bands on the membrane using the available apparatus?

    Thank you for your advice 

    Zaynab Saad

    Thank you for this brief description 

  • Lucas Araújo Caldi Gomes asked a question:
    Open
    Can anyone recommend an anti-p250GAP antibody for western blot?

    I am working with mouse primary midbrain neuronal cultures, would be glad to have an indication on which antibody I should use for p250GAP bloting.

  • Yosif El-Darawish added an answer:
    5
    Why am I getting so many non-specific bands on my western blot?

    I'm getting a ton of non-specific bands in my westerns. 

    My procedure is as follows, and I'm using 25 ug of protein per lane - the bare minimum I think I can get away with. 

    1. Ponceau S stain

    2. Wash 3x 5 mins + 1x 15 mins in TBST

    3. Block in 5% BSA in TBST

    4. Wash 3x 5 mins + 1x 15 mins in TBST

    5. Incubate in Primary 1 hr at 4 degrees (1:1000 in 1% BSA in TBST)

    6. Wash 3x 5 mins + 1x 15 mins in TBST

    7. Incubate in secondary 1 hr at 25 degrees (1:10,000 in 1% BSA in TBST)

    8. Wash 3x 5 mins + 1x 15 mins in TBST

    9. Detect.

    The antibodies that I'm using are relatively new, and have produced clean blots in the past. The problem with the non-specific bands comes and goes, seemingly without rhyme or reason. 

    My primary antibodies target JNK, phospho JNK Thr183, PKC alpha, phospho PKC alpha Ser657, STAT3, and phospho STAT3Y705. 

    Lysates were prepared in either invitrogen Cell Extraction buffer supplemented with pierce protease inhibitor tablets and PMSF, or in CelLytic M with pierce protease inhibitor tablets and Sigma Phosphatase Inhibitor Cocktails 2/3 at 1% v/v. Samples were prepared with freshly made 5x sample buffer, and were heated at 95C for 5 mins immediately prior to loading. The problem occurs in a fashion seemingly independent of sample collection procedures. 

    I've attached pictures of a few problem blots. Your suggestions are greatly appreciated. 

    Yosif El-Darawish

    I found this guide guide to be quite helpful. See the attached file

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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