Western Blot

Western Blot

  • Eiji Kinoshita added an answer:
    Accuracy of Phos-tag gels
    Is anyone working with Phos-tag gels? I have just started out working with them and I understand that with such gels, the protein marker is not an accurate indication of the weight of the protein of interest. However, after blotting for my protein interest, it seems that the darkest bands seen are in the range of 25- 35kDa where in theory, it should have been seen at around 60 - 70kDa. Is this some non specific binding of the primary Ab or is there more to the phos tag gel that I do not know about.
    Eiji Kinoshita · Hiroshima University

    Hi, Joshua.

    I think you should optimize the concentration of acrylamide.
    I recommend using ~12.5% (w/v) polyacrylamide gel for low-molecular-mass proteins of ~17 kDa.
    As another strategy, I have used a precast Phos-tag gel, SuperSep Phos-tag, which is commercially available from Wako, for analyzing a smaller protein, histone H3, ~15 kDa. The precast gel is a 12.5% (w/v) polyacrylamide gel containing immobilized Zn(II)–Phos-tag (50 µM). Although no information on the neutral-pH buffer system used in the precast gel is provided, the manufacturer recommends the use of a general Laemmli’s electrophoretic running buffer consisting of 25 mM Tris, 192 mM glycine, and 0.10% (w/v) SDS (Tris–glycine buffer). I examined the potential usage of a Tris–Tricine buffer (50 mM Tris, 50 mM Tricine, and 0.10% (w/v) SDS) as an alternative running buffer for the SuperSep Phos-tag precast gel system in the analysis of low-molecular-mass phosphoproteins. Compared with Tris–glycine, the Tris–Tricine running buffer improved the resolution of 8.8–35 kDa phosphoproteins and phosphopeptides. Thus, use of this good tool is worthy of consideration for a laborsaving, timesaving, and substantial screening in the reliable detection of low-molecular-mass phosphoproteins. I attached our data file for the analysis of histone H3 and our paper on SuperSep Phos-tag.

    Good luck!

  • Sobia Ejaz added an answer:
    Is my protein sample denatured or is there any contamination?

    Initially when I ran sds-page of my protein sample It gave many bands (around 10 bands in a lane) but now when I did SDS of the same sample it gave fewer bands than before (3-4 bands). Why is it so? Moreover, it showed smeared bands. Is my sample degraded or is there any contamination?

    Sobia Ejaz · H.E.J. Research Institute of Chemistry

    Thank you all for your suggestions. Soon I'll follow these tips.

  • Jan Piwowarski added an answer:
    Which are the best conditions for tranfering a 65KDa protein in western blot?

    to Nitrocellulose:

    a) gel % (8 or t10%)

    b) time and voltage tranference (10V overnight or 20V maybe)

    I've tried tranference 100V to 1h and did not work, however tranference overnight is suitable but i'm having problems with the tranference, after the transference I did coomasie blue stain to the gel and most of the little band transfer ok but not the one i'm looking for.

    Jan Piwowarski · Polish Academy of Sciences

    Please tell me your transfer buffer composition and the model of the transfer apparatus. Maybe there is an answerr to your question...

    Regards - jan

  • Rajesh Rajaiah added an answer:
    What are the best antibodies for MMP- 2 and -9 western blot in CD34+ cells from peripheral blood?

    I need to measure the MMP-2 and 9 protein levels and active form in CD34+ cells cultivated in semi-solid media.

    What are the best antibodies?

    Rajesh Rajaiah · U.S. Food and Drug Administration

    Can you add minimal PBS and spin your semisolid media? If you can, then you can measure MMP2 and MMP9 activity. Activity gel is very sensitive, they can cleave gelatin even at pg level.

  • Valeria De Arcangelis added an answer:
    How do you get a band in a western blot for Insulin receptor beta and phosphorylated insulin receptor beta?

    I have done my western blotting using C2C12 cell lysate sample but I am not able to get the band for the above.

    I am using Santa Cruz antibodies for the insulin receptor beta subunit and its phosphorylated form.

    Valeria De Arcangelis · Catholic University, Rome Italy

    Hi, you already verify that your stimulation work well, the only thing that it is strange is that you don't have the signal not only of phopshorylated receptor but also of non-phopshorylated one. If the Santacruz datasheet assure you the detection in mouse cell lysates (with an image), you can contact the Santacruz support desk, they help me with an antibody (at the end was so old that I have to buy it again).

    You try with super ECL only to confirm that you didn't reveal anything?

  • Jürgen Denecke added an answer:
    How can I prepare Western blot samples from vesicles?

    I'm going to isolate vesicles from bacterial supernatant and run western to see if my protein was incorporated into them. After ultracentrifugation I obtained pellet which supposedly contain vesicles. Should I resuspend it in PBS with SDS loading buffer, boil and run the gel? 

    Can I quantify protein content prior boiling by nanodrop? In this case how do I break vesicles to release proteins?

    Jürgen Denecke · University of Leeds

    1) First aim should be to detect your protein in the pellet. No need for PBS, I would directly add SDS loading buffer + DTT to the pellet, heat to 95 degrees Celsius for 5 minutes, and load on gel twice, once for coomassie staining or silver staining, the other for western blotting. This will give you a starting point.

    2) If you cannot detect your protein, then you have to rethink everything.

    3) If you can detect your protein, then you have an estimate from the gel staining how many other proteins are present. Then you can try repeating the procedure, but this time you sonicate the pellet with PBS first, measure protein concentration, load a specific amount on the gel and detect with western.

    4) However, to determine if your protein is present in vesicles (from a bacterial supernatant) or in large molecular weight protein aggregates is harder, you would have to do a protease protection experiment. Bacteria usually don't produce vesicles in the supernatant, bacteria don't have a secretory pathway with individual membrane compartments either, so this is either a very innovative line of research, or perhaps there is confusion about the term vesicle.  

  • Aleks Po added an answer:
    What causes actin to "spread over" on Western blot?

    Hello my dear fellows,

    My Beta-actin for Western started to look a little strange. The bands look wide and kind of spread over, instead of being more narrow and compact. I am not sure what causes it.

    I was wondering if anybody has encountered this problem before and how did you go about fixing it

    Thanks!!

    Aleks Po · California State University, Channel Islands

    Hi Valeria,

    I decreased the voltage to 100V and it solved the problem.

    Thanks

  • Aleks Po added an answer:
    Why would Total Tau antibody not work on Western blot?

    Hi Colleagues,

    I just did a western today to look at Phospho Tau and Total Tau protein levels in hippocampus of 3TgAD mice and met some complications

    [Phospho Tau Pierce MN #1020; Total Tau BD556319]

    I didn get any staining for Total Tau!?

    I am trying to figure out why.

    The Tau protein can be present in soluble and insoluble forms. The soluble form is found in the supernatant of the homogenate, and the insoluble Tau in the pellet, dissolved in formic acid.

    [The tissue was homogenized in RIPPA with protease inhibitors (including phosphatases)]

    I run the samples to test for soluble Tau.

    First, I stained for Phospho Tau, got a very strong signal in 3 samples.

    Then probed for Actin, which looks fine, overloaded, but fine

    I, then, stripped the blot with b-me containing buffer (to probe for T Tau) and stained with Ponceau S to make sure there is still some protein left.

    The Ponceau looks fine

    I proceeded to probe for Total Tau, but when I develop the blot I have no signal

    There is a dark spot that comes up from long incubation with ECL. I am not 100% sure what it is, but I think it is just from over exposure of the blot

    I was pretty certain to see something for Total Tau.

    What would cause this?

    I am thinking it is a problem with the primary antibody or maybe it is tihe unfavorable blocking/primary incubation buffers?

    Does anybody ever had this problem and how did you fix it?

    I loaded 40ug of protein. We should be seeing something, I hardly think that there is not enough protein left to be tested

    Any suggestions would be highly appreciated

    Thank you all

    You guys are lifesavers and absolutely the best!!

    P.S.I included some images below

    Aleks Po · California State University, Channel Islands

    The antibody wasnt good. Tried a new one and it did the magic

    Thank you all

  • Sumaiya Nabi added an answer:
    What should be the highest speed when centrifuging cell lysate for CoIP?

    I've done one co-immunoprecipitation where after incubating the antigen to the bead-Ab complex for 12 hours at 4oC with rotation, I took the lysate (what's leftover) from the tube and reserved this later for western blot detection. I found my "prey" protein in that lysate, indicating either the receptors do not form a complex or, if they do, it was disrupted before adding the cell lysate to the bead-Ab pair. After lysing my cells in a dish, I centrifuged them at 5000rpm (4500rcf or 16.1G): is this too high of a speed that it might disrupt the receptors if they do interact?

    Sumaiya Nabi · University of Kashmir

    Dear Matt,

    One thing more, how is the expression of proteins of your choice in the lysates.. if it is not good then go for transfection, it is difficult to catch endogenous interaction...

    regards

  • R. Jagathesh Chandra Bose added an answer:
    What is the minimum amount of cells necessary for membrane fractioning? And do membrane protein extraction kits work?
    We want to isolate the plasma membrane of cultured adipocytes for subsequent Western blotting, but we are finding some variety in the amount of cells necessary to achieve a sufficient sample amount (from just one T-75 flask to even as many as 50-100 million cells).
    What would be the minimum area or number of cells required?

    Also, has anybody experience with membrane protein extraction commercial kits? Are they more reliable or recommendable than the traditional sucrose gradient-ultracentrifuge method?
    R. Jagathesh Chandra Bose · Chung-Ang University

    a simple protocol

    treatment with hypotonic  buffer  mild homogenization 22000 rpm then centrifuge 6000g then remove soup add .25M sucrose again centrifuge and wash twice.

  • Ketil winther Pedersen added an answer:
    Having issues getting primary antibody to bind during Western blots involving exosomes.
    I have been successfully performing Western blots on cell lysates from human glioma tissue using tags for actin, HSP70, HSP90, and others, however, I cannot get the signal to show up when I include lanes with exosomes. Even when I alternate lanes (cell lysate, exosome lysate, etc.) I get nice crisp bands in the cell lanes but not in the exosome ones.

    I have tried Laemmli 2x buffer as well as RIPA buffer. Transferring the proteins from gel to PVDF always works, for both cells and exosomes, and I get clear transfer in exosome lanes at the MW that corresponds to HSP70, which is what I'm most interested in.
    Ketil winther Pedersen · ThermoFischer Scientific,

    Hi Lin, the protocol is also on its way to you too.

    take care

    ketil

  • Sofia Omari added an answer:
    Is there a way to preserve PBMCs for later protein extraction and western blotting?
    I would like to collect PBMCs from patients to later extract protein and do western blotting eventually. I wonder if there is a way to preserve the protein or the cells (in the field), so my samples won't ruin with time as I'm supposed to do a flow cytometry protocol on other samples in the same day.

    I tried to use RNAlater from Life Technologies, as they said I can preserve RNA or protein in it. I put the cells there with 5x the volume of RNAlater, left them in at 4C for 3 days to test. Then I extract the protein and there was no much protein which was confirmed with the western blotting. I got no GAPDH bands.
    Sofia Omari · University of Tasmania

    Hi Santhosh,

    Unfortunately, I did not find a way to preserve the PBMC intact, but instead I made the process shorter by collected the blood using BD Vacutainer®CPT™ Tubes according to manufacturer’s instructions. After PBMCS extraction and washing, I lysed the protein using Sigma Mammalian Cell Lysis kit (MCL-1) (Sigma Aldrich, St. Luis, USA) and assayed the protein using Bio-Rad DC Protein Assay kit.

    The other thing you can do,which I also tried, is to collect the washed cells in an Eppendorf tube and remove any excess fluid around them as the water crystal will scratch and lyse the cells, then deep freeze them immediately for later process. However, you may loose some of the protein.

    Hope this will help!

  • Rini Yanti added an answer:
    Do these bands separate well or do I need to re-run the samples with another method?

    There are same thick bands of unknown protein. I'm not sure whether this band because of containing of salt or because of the low of molecular weight. I need some discussion or sharing experience from you. Thanks a lot ..

    Rini Yanti · Gadjah Mada University

    ya Vivek, i already tried  the higher percentage, i run 15 % last friday, and i got 9 bands...  Only 1 thick, that i have now. Today i try to run the sample with 17% polyacrilamide

  • Jiahui Wu added an answer:
    Does anyone have the experience of doing the western blot of extra matrix proteins (glycoproteins) such as nidogens, laminins and collagens?

    I've been trying to detect the proteins indicated in the title in rabbit cornea and cultured rabbit fibroblast cells. For rabbit corneas samples, I use 6M urea to dissolve the cornea; for rabbit fibroblast cells, RIPA buffer was used to lyse the cells. The primary antibodies were all against human (from santa cruz) because we cannot find commercial antibodies for rabbits. I tried 8%/6%/sequencing and tris-acetate gel, semi-dry/wet transfer and different ab concentrations, but I can still not detect any bands, and the background is very high. However when we use the same ab to do immunohistochemistry to rabbit cornea tissues, we can see the fluorescence protein. So could anyone give me some suggestions about the western blotting? Is it because of the protein extractions or other steps? Thank you very much in advance!

    Jiahui Wu · Cleveland Clinic

    Hi Ana-Maria,

    I got this work done couple of months ago, I just want to share with you the method I used. As you suggested, I tried to scraped the proteins but it did not work. Maybe the concentration is too low. I followed one paper, they added 4M guanidine solution to the cell plates to extract the total proteins, including the cell lysate and ECM proteins. After that, the plate surface was scraped and the solution was dialysis exhausively in water. The water-insoluble fractions were collected and re-disovled in 6M urea for western. Most of the ECM proteins are in water-insoluble, and this method was working very well for my protein detection such as nidogen-2. I hope it will be useful for you. Thank you.

  • Arvind Singh Chandel added an answer:
    Does anyone have seen several bands on a blot when checking for Mfn2 in humans cells?

    Hej!

    I am currently working with Mitofusin-2 protein and I have been using an antibody from Sigma that binds to the N-terminal of the protein and most of the times I see only one band on my WB but once in a while a bunch of bands decide to appear on my membrane. I have read that the protein can be ubiquinated (which explains the upper membranes above the normal MW of the protein) but I cant understand what are the band bellow the 86kDa of the protein.

    Does anyone know any paper or have had the same experience as me that could explain this band with a lower molecular weight that the original protein)?

    Arvind Singh Chandel · Central Salt and Marine Chemicals Research Institute

    hello dear Nuno Adam B Shapiro sir is absolutely right on your question 

  • Patrick Aouad added an answer:
    Is there any problem in incubating a Western blot membrane together with more than one antibody?

    I need to see 3 different proteins of different sizes (about 20 kDa of difference between them) and I want to know if it is possible to see all of them at the same time or it is necessary to incubate antibodies sequentially.

    Patrick Aouad · American University of Beirut

    Hi Laura,

    Based on your molecular marker, you can cut the membrane into 3 and stain each with the corresponding antibody. that way you are minimizing the background noise and all the non-specific binding of your antibodies.

    However, if your antibodies are good and their secondary is the same (i.e. all rabbit, or mouse), then I don't see any problem with incubating them all together.

    Good luck!

    Patrick 

  • Ruairidh Edwards added an answer:
    How can only a long isoform of a protein be expressed and not the short?

    I might be over thinking this but my problem lies with understanding how only a long protein isoform can be expressed when the short isoform is encoded by the same region, just 85 amino acids shorter. When I carry out a western blot using 4 different antibodies (2 of which can detect both isoforms) the only isoform to show up is the long isoform. A qPCR was then carried out using isoform 2 specific primers which showed no expression at all. Could somebody explain to me how this happens or possibly point me in the right direction?

    Ruairidh Edwards · University of Dundee

    Thank you very much for the comments.  Sorry I cant answer your question, it wasn't me who designed the primers. My colleague made the primers.

  • Jianbo Jia added an answer:
    What is the best lysis buffer in extracting the total proteins of mouse liver for Western Blot?

    I want to extract the total proteins in liver of a mouse for analyzing several proteins, including phosphorated proteins. What is the best lysis buffer? It would be better if there were reagents protecting phosphorated proteins in the lysis buffer.

    Jianbo Jia · Shandong University

    Hi Huan. Thanks a lot for your suggestions. They are useful for me.

  • Kay-Dietrich Wagner added an answer:
    What is the best way to strip a Western Blot?

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

    Kay-Dietrich Wagner · University of Nice-Sophia Antipolis

    We use Antibody stripping buffer from Euromedex. It is relatively mild, cheap, and without Mercapto. Best is to start with your protein of interest and do the strongly expressed Housekeeping's afterwards. Or as mentioned above, if you have tons of material, load several lanes and cut.

  • Arkadiusz Kozubek added an answer:
    Why should we run SDS-PAGE on constant voltage and Western Blot transfer on constant current ?

    Protocols disagree on the  exact voltage and current set up for the above technique

    Arkadiusz Kozubek · University of Wroclaw

    I just try to sumarize the discussion as

    For electrophoresis relationships key electrical parameters are expressed in two basic formulas:
    The first is the Ohm's law, I = U / R, stating that the electric current (I, the intensity, in Amps or Milliamps) is directly proportional to the voltage (U, Volts) and inversely proportional to the resistance (R, Ohms). The second is the relationship P = U x I saying that the power (P, watts), a parameter indicating the quantity the heat generated during flow of the current, so depending on both the voltage and the current. It also depends on the resistance (P = I2 x R). When one of the three parameters is kept constant the others will change. Typically electrophoresis process is conducted while keeping constant the value of current, voltage or power. In most processes, the resistance of the gel increases with the progress of separation. This increase causes changes depending on the parameter electrophoresis maintained at a constant level. And will result in heat generation.
    During electrophoresis, preventing excessive temperature rise is the key to avoid e.g. denaturation of some of the proteins from the sample. The gels plate, even in the technique applying the denaturation of proteins (SDS-PAGE), the excess of heat results in a temperature gradient between the edges and the center which results in an uneven migration of sample components i.e. “smiling”. The middle bands migrate slightly faster than the bands located at the edges of the gel, which causes difficulties in the analysis of the results section. Getting "smiled" gels is not an inherent feature of different designs of electrophoresis apparatus. Even the apparatus from well-known and reputable company can produce so disturbed separations when to high current is applied (I = const at to high level) and when the heat dissipation system can not cope with so much of heat. On the other hand, even a simple apparatus would provide ideal separations, if too high current is not used. Ideally 20-25o centigrade should be maintained during electrophoresis.
    Therefore solutions should be (in my opinion) – constant power or constant voltage. The last will prolong the sepatation but will restrict generation of heat excess as the current will drop gradually as the result of the increase of gel resistance. But if you keep I=const and you will result in smiling, just decrease current settings.

  • Cheng-Xin Gong added an answer:
    Is there any marker for neuronal necrosis that you can see on western blots as cleaved caspase for apoptosis?
    We'd like to study the time courses of neuronal apoptosis vs. necrosis in the mouse brain and prefer to use western blots than morphological methods. Does anyone know such a marker for necrosis?
    Cheng-Xin Gong · New York State Institute for Basic Research in Developmental Disabilities

    Many thanks, Praneeti.

  • Carlton Hoyt added an answer:
    Does anyone have any suggestions for the best way to homogenize brain tissue for western blot?
    I have been using a polytron, but perhaps there is a better way.
    Carlton Hoyt · BioBM Consulting

    If you're using an automated homogenizer (rotor-stator, ultrasonic, bead mill, etc.) you're going to get denaturation.  It's practically unavoidable.  If your analysis depends on retaining native state proteins, you'll probably want to stick with a manual dounce homogenizer.  This will minimize foaming, cavitation, vortexing, and creation of liquid-air interfaces more generally.

    If you're not worried about the proteins being in the native state, any type of homogenization can be used.  Brain tissue homogenizes very easily.  The consideration should then be based more on sample size, throughput, and projected future needs.

    If you would like, you can learn more about different homogenization technologies here: http://homogenizers.net/pages/ac-application-center

  • Rina Bandopadhyay added an answer:
    What Human cell lines express alpha-synuclein for RT-PCR primer validation?

    We recently ordered several primers for mRNA detection of human alpha-synuclein by RT-PCR, and I'm trying to find a cell line for validation. I've found publications that report detection of human alpha-synuclein by Western Blot in SH-SY5Y and HEK293 cells, although the latter surprises me. Does anyone know if these would be suitable for my purposes?

    Rina Bandopadhyay · University of London

    SH-SY5Y cells will be suitable.

  • Sagarika Utture added an answer:
    Could anyone please suggest a good positive control for western blot...I am trying to detect cfos and cmyc proteins?

    I have extracted proteins from rat heart tissues and performed a western blot analysis on the same. Used cmyc antibody and cfos antibody purchased from Santa Cruz Biotechnology. Bands are detected but not at the proper band size. The control- Actin shows up properly in every single experiment performed. 

    Sagarika Utture · Brandeis University

    Thank you for all your inputs..I will try altering the parameters according to suggestions and see how the samples run.

    @ Hardik thanks for the tip ..will call the company and have a discussion about the same.

  • Maathangi Murali added an answer:
    Why am I getting horizontal streaks in 2D gels?

    Dear All,

    I am doing my 2D electrophoresis. I had run duplicate sample for all the protein and got these two gels. I have run first dimension using 3-10pH Nolinear 17cm BioRad strips. But both gels are too different. Everything is same for both gels during first dimension. The only difference in second dimension is that HB02R_T10001 was run and stained on first day while HB02R_T20001 was run and stained on second day. The gel was 12.5% acrylamide and 1X trisglycine + 2% SDS was used in running buffer. There is too much horizontal streaking in first gel while the second one is quite good. This does not happen to me before. High molecular weight proteins are not well separated. Moreover, if any one can tell me how to know the protein sample is degraded because it gives good quantification results. I am waiting for reply.

    Maathangi Murali · Art of Living Research

     loading in the gel might not be proper. U can check the voltage used for electrophoresis

  • Shenq-Shyang Huang added an answer:
    Have I obtained the optimal micrograms of IP antibody for co-immunoprecipitation assay?

    I am conducting a Co-IP with two receptors (one viral one human). Very large, nonspecific, and heavy bands appear for the Co-IP samples in a western blot twice. I've used 5ug and 10ug of IP antibody. Is this too much IP antibody? Also, positive control bands (lanes on right) don't appear in the WB when added with the co-IP samples (lanes on left), but when troubleshooting to see what is the best lysis buffer to use, they do appear. Are those large and heavy bands (from CoIP samples) affecting the appearance of my positive controls? 

    Any advice will be greatly appreciated

    Shenq-Shyang Huang · National Health Research Institutes

    I optimal incubation time is between 16-20 hours at 4oC with rotation. Longer incubation time may cause protein aggregation.

  • Shang-Feng Gao added an answer:
    What is the best stripping solution recipe (in house) and method for Nitrocellulose and PVDF membrane?
    A) Do you agree this is the best stripping solution recipe :

    Preparation of 100 ml Stripping Solutions (Final Concentration = 100mM):
     2 gm SDS
     6.25 ml 1M TRIS [PH=6.7]
     Make it up-to 100 ml with water (Millipore)
     Add 704.2 µL Beta-Mercaptoethanol

    B) Please share the method you use for stripping the membrane i.e. incubation time period and condition

    After stripping the membrane, I would like to reprob the membrane with another antibody. For e.g. Probing the membrane first with p-ERK1/2 and latter reprob with ERK1/2 primary antibody.

    Thanks a lot for your expertise opinions.
    Shang-Feng Gao · Xuzhou Medical College

    I have the same question too. Does anyone have the experience to prepare the stripping buffer in house?

  • Lorna Cryan added an answer:
    What is a good antibody for phospho serine/threonine?

    Could anyone recommend a good antibody for western blotting for global analysis of changes in phospho-serine and threonines?

    There are many available from various vendors (e.g http://www.cellsignal.com/product/productDetail.jsp?productId=9631) - any personal experience of one working well?

    I believe these can be trickier to work with than phospho-tyrosine (4G10) antibodies.

    Cheers!

    Lorna Cryan · Harvard Medical School

    Thanks for your thoughts.  That was what I was thinking.  We may need a more sensitive method such as SILAC + TiO2 enrichment.

  • Nisha Rizvi added an answer:
    Problem isolating biotinylated proteins from brain tissue - can anyone help?
    I would like to isolate exclusively membrane expressed proteins from rat brain tissue (thalamus slices 200 microns) and run a western blot. I tried to use different available biotin as well as commercial available kit for biotinylation and subsequently isolation of biotinylated protein. However, I have not succeeded in it.
    Commercial available kit has Neutravidin columns and to elute protein we need to use DTT 50 mM. Then to measure the correct protein concentration we have used reducing agent compatible protein assay, but acceptable concentration of reducing agent in given assay is 5 mM, so we dilute the samples 10 times, after diluting we cannot even detect protein in our sample (protein sample was too much diluted?)
    When I tried to use streptavidin beads it was very difficult to isolate protein
    Nisha Rizvi · Southern Illinois University School of Medicine

    Hi Tushar,

    I am trying to do the same as you. Isolate only CELL SURFACE proteins (avoid contamination from any cytoplasmic material including organelle membranes)  in rat hippocampal tissue. Did you figure out how to do it? I saw a JOVE article but before I start, wanted to check other protocols. Any thoughts?

    Thanks

    Nisha

  • Jordi Pou Sánchez added an answer:
    How does current interruption affect Western Blotting?

    While I was blotting after 3-4 hours electricity went down during night and I didn't realize. After 20 hours I looked at the blot, marker was already slightly seen on the membrane but also some on the gel. I put blot on again. Do the protein samples dispearse or are washed away while there is no voltage/current running? Does it affect the blotting process? Will there be any protein left on the membrane?

    Thanks for helping.

    Jordi Pou Sánchez · IMIM Hospital del Mar Medical Research Institute

    Hi Elena,

    As everybody pointed you, although transfer is highly dependant on the voltage and the type of blotting (wet or semi dry), the time of transfer you used should be enough for having good yield of protein on your membrane. I recommend you try shorter times in the future, in order to avoid some problems as difusion of the samples due to interruption of the voltage (I usually use 90-100 minutes, 200mA for wet transfer and it works perfectly).

    But, anyway, if you have detected bands in Ponceau staining, it's a happy ending for your WB :)

    Good luck in the future!

    Jordi

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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