Western Blot

Western Blot

  • Ariel Orbach added an answer:
    3
    How much voltage/current should I set to transfer minigel in Western blot?

    Is there any scale for setting voltage/current in running Western blot transfer? Is it different when using different sizes of gel?

    Ariel Orbach

    If you hare using wet transfer (i.g. Biorad) it should run on 400mA (constant) for 1 hour (keep cold). However, today there are devices that enable rapid transfer (less than 10 minutes).

    Good luck.

  • Deo Singh added an answer:
    1
    Can anyone help me with ASC oligomerization assay?

    I have tried to detect ASC oligomers after overexpression but still struggling.

    I can see ASC monomers and just smears somewhere above 100 kDa, but not dimers, trimers and oligomers in WB.
    Any suggestions for troubleshooting?
    Here are my procedures.

    293T cells (10^6 per well on a 6-well plate) were transfected with 1 microgram of each plasmid of NLRP3 and ASC (expression of tagged proteins were confirmed).
    For ASC oligomerization by crosslinking,
    1. Wash the cells and lyse the cells with 100 microliter of Buffer A.
    2. Shear the lysates with 20 times through 27 G needle.
    3. Centrifuge at 340 g for 8 min at 4 °C.
    4. Collect supernatant and add 1 vol. CHAPS buffer
    5. Centrifuge at 2650 g for 8 min at 4 °C.
    6. Resuspend the pellet in 20 microliter of CHAPS buffer containing 10 mM BS3 at room temperature for 30 min.
    7. Add 5X SDS sample buffer and boil.
    Then, Western blot with a 10% gel.

    The assay was done according to this reference (except for the use of different crosslinking reagent).
    https://www.researchgate.net/publication/271223869_Syk_is_involved_in_NLRP3_inflammasome-mediated_caspase-1_activation_through_adaptor_ASC_phosphorylation_and_enhanced_oligomerization


    Buffer A and CHAPS buffer were prepared according to this reference.
    https://www.researchgate.net/publication/251452825_Chapter_Thirteen_Assembly_Purification_and_Assay_of_the_Activity_of_the_ASC_Pyroptosome

    I've also done based on this paper, but in vain.
    https://www.researchgate.net/publication/264988115_Influenza_A_virus_protein_PB1-F2_translocates_into_mitochondria_via_Tom40_channels_and_impairs_innate_immunity

    • Source
      [Show abstract] [Hide abstract]
      ABSTRACT: NLRP3 is the most crucial member of the NLR family, as it detects the existence of pathogen invasion and self-derived molecules associated with cellular damage. Several studies have reported that excessive NLRP3 inflammasome-mediated caspase-1 activation is a key factor in the development of diseases. Recent studies have reported that Syk is involved in pathogen-induced NLRP3 inflammasome activation; however, the detailed mechanism linking Syk to NLRP3 inflammasome remains unclear. In this study, we showed that Syk mediates NLRP3 stimuli-induced processing of procaspase-1 and the consequent activation of caspase-1. Moreover, the kinase activity of Syk is required to potentiate caspase-1 activation in a reconstituted NLRP3 inflammasome system in HEK293T cells. The adaptor protein ASC bridges NLRP3 with the effector protein caspase-1. Herein, we find that Syk can associate directly with ASC and NLRP3 by its kinase domain but interact indirectly with procaspase-1. Syk can phosphorylate ASC at Y146 and Y187 residues, and the phosphorylation of both residues is critical to enhance ASC oligomerization and the recruitment of procaspase-1. Together, our results reveal a new molecular pathway through which Syk promotes NLRP3 inflammasome formation, resulting from the phosphorylation of ASC. Thus, the control of Syk activity might be effective to modulate NLRP3 inflammasome activation and treat NLRP3-related immune diseases. © Society for Leukocyte Biology.
      Full-text · Article · Jan 2015 · Journal of Leukocyte Biology

    + 2 more attachments

    Deo Singh

    Dear Hong Su,

    You needed to use biophysical methods to study oligomerization. Cross-linking is not very good method to study oligomerization. Use FRET to study it. But you needed to be very careful.

  • Ana Maria Calderon added an answer:
    1
    Can I check the specificity between glycan and Lectin as below design?

    Firsty, I found that  fucosylation level of anti-A protein antibody is significantly lower than anti-B protein antibody with ELISA using lectin. (Coating: A or B protein, Blocking, Sera, Biotinylated AAL, HRP conjugated streptavidin)

    And I wanna confirm this Data, but it is so difficult to confirm with Lectin blotting. So I have an idea that could instead lectin blotting, that is firstly I would purify fucosylated substance from serum by AAL agarose resin and check the antibody level of anti-A and anti-B using ELISA. (Coating: A or B protein, Blocking, fractions from AAL agarose chromatography, HRP conjugated anti-human IgG) So I could check if the antibody level of A is significantly lower than B. 

    Can anyone advise me whether the design is reliable?

    Thanks so much. 

    Ana Maria Calderon

    Your description is so confusing for me. Are you looking for a fucosylated substance in serum which interfere with the antigen-antibody affinity? what about if the A or B antibodies are more or less glycosilated themselves?. 

    Apparently in your first assay you are not measuring antibody levels but detecting some fucosylation in antibodies. What was your objective, why a lectin was introduced at the assay?.

  • Michael David Hoos asked a question:
    Open
    Does anyone know of an antibody that is specific for the GOLLI region of GOLLI-MBP proteins?

    I need to distinguish GOLLI proteins from their related myelin basic protein isoforms.  It seems that most GOLLI-MBP antibodies are specific for the MBP containing regions of the proteins and thus, it is difficult to tell these apart.  There are so many splice isoforms of this protein family that molecular weight is not sufficient to identify different forms on a western.  I need an antibody that can directly label the GOLLI specific regions of these proteins.  Does anyone know of any?

  • William Virgil Brown added an answer:
    4
    How should you extract protein from tissue that has a high lipid content (such as adipose or fatty liver)?

    Are there any major protocol modifications needed? Could the high lipid content cause problems when Western blotting?

    I was thinking that when you spin the tissue homogenates you can remove or avoid the fatty layer on top. Or you can perform some sort of lipid extraction (such as a Folch extraction). However I am concerned that you couild lose proteins that associate with the cellular lipids.

    William Virgil Brown

    The technique that works best for triglyceride rich lipoproteins is to remove all water with lyophilization (freeze -dry).  Treat the residual with ethanol-ether (2:1) until a dry powder is left and solubilize in decyl-sulfate (not dodecyl).  You may need about 0.1 M to fully solubilize but you can adjust the concentration of the decyl'sulfate by dialysis.  Do-decyl and many other detergents tend to form micelles and will not dialyes easily. 

    Folsch extraction is too harsh for many proteins.  You may need to use less ethanol and more ether depending on the solubility of the proteins in question in ethanol.

    I hope that helps.

  • David Ferland added an answer:
    4
    How can I isolate the contents of a vesicle?

    I want to be able to look at the contents of a vesicle with both immunofluorescence and western blot. Are there ways to permeablize cells to get an antibody into the vesicle but not burst the vesicle? Are there ways to isolate the vesicles themselves so I can lyse them and analyze their contents?

    David Ferland
    Thank you Maurizio, that is what I meant. I want to look at intracellular vesicles carrying my protein of interest from the golgi to the membrane for secretion. What I was worried about in cell fixation, was that the permeabilization step would disrupt the membrane of the vesicle too much and I wouldn't be able to colocalize a vesicle marker with my protein.

    As for the western blot, I want to see what enzymes are in the vesicles along with my protein of interest. I'll look into centrifugation techniques.
  • Rafik Karaman added an answer:
    4
    Any suggestion of a whole cell western blot protocol from rat testis?

    Dear,

    Anyone has/tried some protocol for extraction of whole cell from rat testis for western blot? Which buffer you used, troubleshooting and similar things.

    Rafik Karaman

    Dear Milos,

    My intention was that you look at the buffer suggested by Sigma for their product.

    Rafik

  • Sumeet Manandhar added an answer:
    4
    Why consistent bands are difficult to get in western?

    I have been doing western blotting for past 8 months. i am not able to get consistence result of my needed protein .by sometimes i get distorted band and sometimes ok and sometimes i don’t get any band at all. but every time i do i have b- actin. i had one question. if my lysate preparation step is wrong do I see b actin band or not? or if sometimes if unknowingly I use cell debris  in cell lysate do I get b actin bands or not?And whether it is due to the degradation of my needed protein in environment? I use protease inhibitor and PMSF and I do all cell lysate preparation in ice. Do you think that something is wrong with my cell lysate preparation?

    Sumeet Manandhar

    Thanks for the reply.Professor Saleh alkarim I do the quantification of protein every time. I load the same amount of sample. I have attached by results below can u evaluate it.

  • Rafik Karaman added an answer:
    1
    Can anyone suggest a rt-pcr kit for detection of p53 and bcl-2 in apoptotic cells?

    Hi everyone!

    Iam searching for a good kit for studying the expression of p53 and bcl-2 in apoptotic cells through rt-pcr and western blotting.Can any of u suggest it.

    thanks.

    Rafik Karaman

    Dear Syed,

    Please see attached file:

    Apoptosis Detection
    Kits
    CYTOC-OX1 Cytochrome c Oxidase Assay Kit For the determination of cytochrome c oxidase activity in mitochondrial soluble and membrane
    Sufficient for 100 tests bound samples. Cytochrome c oxidase [EC 1.9.3.1.] is present in mitochondria of the more
    highly developed cells and in the cytoplasmic membrane of bacteria. This enzyme provides
    energy for the cell by coupling electron transport through the cytochrome chain with the
    process of oxidative phosphorylation. Cytochrome c oxidase is located on the inner mitochondrial
    membrane that divides the mitochondrial matrix from the intermembrane space
    and traditionally it has been used as a marker for this membrane. The colorimetric assay
    measures the decrease in absorbance of ferrocytochrome c caused by its oxidation to
    ferricytochrome c by cytochrome c oxidase.
    PCR Primer Sets
    APO-PCR Apoptosis PCR Bax/Bcl-2 The kit is useful for studying the progress of apoptosis and the evaluation of potential
    Multiplex Primer Sets apoptosis inducers.
    Sufficient for 50 (50 ml) PCR reactions Bcl-2 is a member of a large gene family encoding proteins that can either inhibit (e.g. Bcl-2,
    Bcl-XL ) or promote (e.g. Bax, Bcl-XS, Bak) apoptosis. Bcl-2 inhibits apoptosis by preventing
    the release of apoptosis inducing factor (AIF) and cytochrome c from the mitochondria. If
    the Bcl-2 level is higher than the Bax level, apoptosis will be inhibited. The ratio of Bcl-2
    to Bax in the cell can determine whether or not the cell initiates apoptosis or survives.
    Some cancer cells such as melanoma overexpress Bcl-2 preventing apoptosis and allowing
    malignant growth to continue.
    The kit contains primer sets for the detection of Bax and Bcl reverse transcribed from mRNA
    from rat, mouse or human tissues or cell lines (reverse transcriptase and thermostable
    DNA polymerase not included). A primer set for a “housekeeping” gene, GAPDH, as an
    internal control is included in the kit. These primer sets are optimized for the simultaneous
    amplification, detection and comparison of Bax and Bcl-2 mRNA levels. The sizes of the
    amplified products are 487bp for Bax, 127bp for Bcl-2 and 349bp for GAPDH.
    Features and Benefits:
    • Simultaneous RT-PCR amplification and detection of Bax and Bcl-2 mRNA levels.
    • Includes primers for the GAPDH gene as an internal control for comparison of Bax and
    Bcl-2 mRNA levels to allow for semiquantitative evaluation of Bax and Bcl-2.
    • Functions with Taq DNA polymerase, AccuTaq™ LA DNA polymerase, REDTaq™ DNA
    polymerase, and equivalent DNA polymerases.
    • Primer solutions contain both sense and anti-sense primers of the specific gene for
    convenience.
    • The primers work with cDNA reverse transcribed from RNA (mRNA and total RNA)
    extracted from cells and tissues using various extraction methods.
    • Works in one step RT-PCR procedures.
    • Genomic DNA contamination in the RNA will not be amplified.
    Apoptosis PCR Primer Sets Synthetic oligonucleotide primers for RT-PCR analysis of mRNA expression. Amplifies reverseSufficient
    for 50 reactions transcript mRNAs from human, mouse and rat sources. Set includes an upstream and
    downstream oligonucleotide.
    B 3055 Bad PCR Primer Set
    B 3180 Bak PCR Primer Set
    B 8304 Bax PCR Primer Set
    B 9179 Bcl-2 PCR Primer Set
    F 8425 Fas PCR Primer Set
    F 8300 Fas-Ligand PCR Primer Set

    Hoping this will be helpful,

    Rafik

  • Deo Singh added an answer:
    7
    Do I need to add Sampling buffer and heat it at 70C for 10 minutes before running the blot for a cross-linked lysate?

    Hi, I have cross-linked the E-Cadherins using DTSSP. I am planning to run the western blot experiment to see if it forms higher order oligomers. I am not sure if I should use Sampling buffer and heat it for 10 minutes before loading it in the gel. Can anyone suggest if this step is necessary?

    Deo Singh

    Hi Adam,

    I am attaching the blot so that you can look at it. I used 3-8% NuPAGE tris-acetate gel. Running buffer was also tris-acetate. I followed the thermo-scientific protocol from where it is purchased. I cross-linked it at room temperature for 30 minutes. Also cross-linker was dissolved it water.

    Please look at this paper:

    E-cadherin functions as a cisdimer
    at the cell–cell adhesive
    interface in vivo  published in Nature Structural and Molecular Biology in 1999 by Takeda et al

    Figure 1a. They exactly did the same experiment. Used same cross-linker. Used same concentration of cross-linker as I am doing. However, there cell like was different and their protein was not a fusion protein. Also, they used different cell line. I am using HEK293T.

  • Viktor Y Butnev added an answer:
    4
    How do I elucidate the identity of a band from western blot?

    I've been consistently getting a thick band on my western blot, which is not the size of my protein of interest. However, I'm getting convinced, that this band is not unspecific binding of my antibody, but rather a splice variant or proteolytic fragment of my protein. I would like to purify the protein fragment from a gel and run it on ms/ms to sequence it. Do anyone know how time consuming and costly that would be? Or is there a better solution?

    Thanks!

    Viktor Y Butnev

    Another option you might want to consider is to run western blots with your protein sample using monoclonal and polyclonal antibodies against your protein and to the splice variant(s), closely related family member(s), or proteolytic fragment(s) of your protein (if available). Any differences in those western blots will help you to draw some conclusions and further strategies, and you can save some money over any other approaches.  

  • Jessica Poh added an answer:
    4
    I recently did a Western blot with HS6ST2 antibody and my bands had silts in the middle of all my visualised bands. Can anyone enlighten me on this?

    I used 1:5000 dilution for the HS6ST2 antibody and this image shows 15 seconds of exposure using the Pico reagent. 

    Jessica Poh

    Thank you everyone. I will try again! 

  • Viktor Y Butnev added an answer:
    3
    Thick and thin band in Western blots?

    Everyday I do a western blot and I am left helpless for new things keep on surfacing. Can you please explain looking at my poncean where is my experiment going wrong to give me varied thick and thin western blot bands? I carry out western blot by quantifying it using bradford assay followed by using lysis buffer---95C boiling--- loading 30ug of protein. running on precast gels for 1hr and half at 150V---transfer for 2hrs at 100V--and I look at the poncean and I see this.. Please help me out? Western Blot is taking my sleep away and its frustrating. Thank you 

    Viktor Y Butnev

    I also think there is nothing wrong with your picture. The gel and transfer were good. The higher abundance of some of the proteins leads to more intense and broad bands. Besides some of the proteins might have different post translational modifications like glycosylation, phosphorylation and so on, which might cause the bands to be not so sharp. You can go ahead with your western blot development, everything should be fine. 

  • Nitika Nitika added an answer:
    6
    In Western blotting, beta-actin shows equal expression but alpha tubulin does not, why?

    beta actin and alpha tubulin come from one membrane.

    Nitika Nitika

    It isa lso possible that if you are treating your cells with some chemical then tubulin is getting affected in that condition.

  • Muthusamy Kunnimalaiyaan added an answer:
    7
    Any advice on secondary antibody binding without primary antibodies?

    I am new at Western Blotting so unsure what could be going wrong.

    It looks like my secondary antibody (Cell Signalling Goat Anti-Rabbit-HRP) is binding to the PDVF membrane in the absence of a primary antibody, giving bands around 100-110kD and 75kD.

    I am seeing these bands only in the liver lysate and not in my spleen lysate, which is showing my expected Rabbit Anti-mouse Beta-Actin band if it is added to the filter. I never seen the Beta-Actin band in the liver lysate.

    I am currently using 0.05% Tween20-TBS for the washes and secondary antibody dilution, 3% BSA TBS for the blocking and 3% BSA 0.05% Tween 20 TBS for the for the primary antibody dilution. The antibodies are diluted to 1:1000.

    Can anyone help me figure out what could be going wrong?

    Muthusamy Kunnimalaiyaan

    Nice western blot.  Increase BSA to 5% or use 5% milk to block the blot. this might help to reduce the background band (120Kd)

  • Katherine L Wilson added an answer:
    5
    Any advice on Lamin B1 as nuclear control but with no bands shown after WB?

    Hi, all,

    I used lamin B1 (68 kd) as my nuclear protein control since my target protein is kind of large (130 kd). I separated the nuclear protein from cytoplasm using NE-PER kit, then run the WB. My target showed up but blank in my control protein.

    I am using CST Lamin B1 (http://www.cellsignal.com/products/primary-antibodies/lamin-b1-d9v6h-rabbit-mab/13435?N=4294956287&Ntt=Lamin+B1&fromPage=plp) The website said 100% homology to bovine. I used couple of other antibodies like this case worked well. But I never experienced no bands at all for control.

    So I wonder if the problem is the antibody (though 100% homology still not fit for bovine)? or any possible procedure issues?

    I used 1 to 2000 dilution in 5% nonfat milk TBST 4c overnight. The protein level is 20 ug (nuclear part, supposed to be concentrated)

    Thanks for your helps!

    Katherine L Wilson

    Nice discussion. Sonication is required to solubilize >90% of B-type lamins (otherwise they pellet with chromatin), and variable percentages of A-type lamins and nuclear membrane proteins. Also, specific lamins (A, C, B1, B2) are expressed at different levels in specific cell types. Extreme cases, e.g., neurons, express no lamin A, only the shorter isoform lamin C.

  • Joshua Samsoondar added an answer:
    1
    Image Studio Lite vs ImageJ for analyzing western blots: Which one do you prefer? and Why?

    Hello,

    Both are free. From what I have tried,

    1. Image Studio Lite from Licor is much easier to use, but it doesn't have lane background subtraction, which I think is the best method for background subtraction.

    2. On the other hand, ImageJ has more tools to do with, but if I mistake a step (eg. cropping band) I have to repeat all the steps.  Also if the band is very faint or almost no band is very hard to select area under the graph.

    Please share what you think  about these two programs.

    Thank you

    Joshua Samsoondar

    Good question, I have used both for Western blots a fair bit, especially since our lab started using the Li-cor Odyssey Fc.

    ImageJ is very powerful because of the many tools available for it, but for simply doing Western blot quantitation, I prefer Image Studio Lite.

    Image Studio is very good for efficiently analyzing your blots but still allowing you to adjust your calculation parameters (if you're interested). The point and click tool for drawing boxes is very fast but I'm not sure I fully trust it to accurately trace my bands so I prefer to draw the boxes myself. It is also possible to look at the histogram density plots like you would in ImageJ. Overall it's fairly intuitive to use but I would just add that the UI could be improved and some of the extra features don't work particularly well (such as annotations, charts, normalization and overall navigation). Simple quantitations are straightforward but most of the nonessential features are not as user-friendly so hopefully this is improved in newer versions.

    Also, I do believe you can do something similar to a background lane subtraction in Image Studio. In the background tab, select either Average or Median then set the "Segment" to "Top/Bottom". This uses the background values from above and below each box, so each box gets its own own background value (similar to looking a lane background).

    Hope this helps.

  • Timothy J Seabrook added an answer:
    5
    In freezing brain tissue for WB - do you need sucrose?

    Hi all, 

    In my current lab they dump freshly isolated brain tissue into 0.4 M sucrose before freezing on dry ice. Is the sucrose necessary at all when I only want to do WBs i.e. could you get equivalent results by leaving it out? I want to detect protein phosphorylation so I strive to keep the tissue frozen as long as possible before synaptosome preparation and with sucrose I need to thaw the tissue more then if I'd leave it out.

    Best wishes,

    Tau  

    Timothy J Seabrook

    I have run WBs, including for protein phosphorylation, using brain tissue directly frozen in liquid nitrogen. If a crude protein isolate, with subsequent WB, is all you plan to do than sucrose is not necessary. However, if you want to look at proteins in synaptosomes, then sucrose is likely required. 

  • Ilya Tsyrlov added an answer:
    4
    Why in western blot, Ah receptor band is not integrated?

    In the range of 100 kDa ahr with two bands that are very close to each other, can be seen. While the molecular weight of the protein of 100 kD listed.

    Ilya Tsyrlov

    Hi Amir,

    In terms of other colleagues’ comments, please keep in mind that cyanogen bromide fragmentation revealed that both forms mentioned contain the same size N-terminal fragment. Sucrose density gradient analysis of HeLa cell cytosol indicated that both forms cosedimented at 9S. Both the 106 and 104 kDa AhR bands were detected in four different human cell lines. Together, these data would indicate that the AhR in human cell lines exists in two distinct forms.

    Here, let me turn to the major point, namely to the very human cell line you’ve chosen to evaluate the Ah receptor’ physic-chemical parameters. It seems for you are running uphill tough target, because in 2013 and 2014 a team of G. Goode and others demonstrated AhR knockdown in the MDA-MB-231 breast cancer cell line. The latter paper entitled: “Depletion of the Aryl Hydrocarbon Receptor in MDA-MB-231 Human Breast Cancer Cells Altered the Expression of Genes in Key Regulatory Pathways of Cancer”, published in PLoS One. 2014; 9(6): e100103. Published online 2014 Jun 16. doi:  10.1371/journal.pone.0100103. Some details might be revealed from the beneath Abstract: The aryl hydrocarbon receptor (AhR), a transcription factor that is best known for its role in mediating the toxic responses elicited by poly aromatic hydrocarbons as well as many other environmental factors; is also involved in breast cancer progression. We previously reported that stable knockdown of AhR decreased the tumorigenic properties of the highly metastatic MDA-MB-231 breast cancer cell line; whereas ectopic overexpression of AhR was sufficient to transform immortalized human mammary epithelial cells to exhibit malignant phenotypes. In the present study we investigated the genes that are differentially regulated by AhR and are controlling cellular processes linked to breast cancer. We used Affymetrix Human GeneChip 1.0-ST whole transcriptome arrays to analyze alterations of gene expression resulting from stable AhR knockdown in the MDA-MB-231 breast cancer cell line. The expression of 144 genes was significantly altered with a ≥2.0-fold change and a multiple test corrected p-value ≤0.05, as a result of AhR knockdown. We demonstrate that AhR knockdown alters the expression of several genes known to be linked to cancer. These genes include those involved in tryptophan metabolism (KYNU), cell growth (MUC1 and IL8), cell survival (BIRC3 and BCL3), cell migration and invasion (S100A4 and ABI3), multi-drug resistance (ABCC3) and angiogenesis (VEGFA and CCL2). The identification of the genes and pathways affected by AhR depletion provides new insight into possible molecular events that could explain the reported phenotypic changes. In conclusion AhR knockdown alters the expression of genes known to enhance or inhibit cancer progression; tipping the balance towards a state that counteracts tumor progression.

    Best wishes,

    Ilya

  • Cole Peters added an answer:
    5
    Have anyone tried IL-1 alpha antibody for Western blotting by using mouse brain tissue samples?

    Have anyone tried IL-1alpha antibody for western blotting using mouse tissue samples? I have used abcam Il-1 alpha. As per the information sheet two bands were reported (one at 17 kDa and other at 23 kDa). However i found only one band at 17 kDa. What could be the possible reason? 

    Cole Peters

    So you don't need to perfuse unless you really don't want the blood in your samples. After you sacrifice your mice just harvest the brains and mince them in a RIPA buffer supplemented with protease inhibitors.

    Side note, are you sure your antibody is for full and cleaved IL1? You are running your samples in a SDS based loading dye AFTER boiling for 5min? In other words do you have a positive control for full length IL1, to make sure its not an antibody problem?

    If this doesn't work I'd reach out to the company the Ab comes from and authors from papers that looked at IL1 in brain tissues( if any)

  • Grégory Minguet asked a question:
    Open
    Is there any alternative to western blot to assess PCK activities within human neutrophil lysates ?

    -

  • Laura Robrahn added an answer:
    5
    Can anybody help with mTOR Western Blot antibodies and conditions?

    Hello everyone,

    we are currently trying to establish methods to further characterize new mTOR knockout mice. Since we could not find a working antibody as well as conditions for Western Blot and/or IHC, your advice on this would be greatly appreciated!

    Thank you,

    Laura

    Laura Robrahn

    Hi everyone,

    thank you very much for your advice. For now, we ordered the SCBT antibody as recommended by Mark Livingstone and will of course troubleshoot the conditions; protein concentration, ab-concentration... as usual. But thank you very much for your detailed answer Kahlid, we were basically using the same protocol, and since you also did not mention a short half-life, I guess the change of antibody - and depending on the new results, maybe change of detection substrates as you mentioned - could already be sufficient. Assuming that mTOR levels should be relatively high, we only want to see whether the liver specific knockout works, but certainly looking at mTOR signaling is also a good idea to confirm!

  • Mark Bond added an answer:
    5
    Can you import TIFFs into BIORAD ImageLab software?

    We recently got a new BioRad digital imaging western blot detection system. All is fine except that the software (ImageLab) can export images to TIFF but then refuses to import them again for densitometry analysis. Am I doing something wrong! Surely if it can export a TIFF it should also import a TIFF?

    Mark Bond

    No. Its odd that you cant. I think they want you to use their own proprietry .scn format that is a 12bit raw file. More info in this file so better for densitometry. 

  • Yasir Alhamdi added an answer:
    3
    Western blotting for extracellular receptors?

    Hello! I am wishing to a western blot for a protein that is expressed extracelluarly in an adherent cell line. Is it ok to use trypsin when I collect my cells? Or will it destroy the protein? Should I use a dissociation buffer?  Thank you!

    Yasir Alhamdi

    If I understand the question correctly, you are looking for a protein that is RELEASED by your cell line extracellularly? if that is the case, then all you need to do is collect the culture medium after certain period of culturing the cells and look for your protein in the media. Collecting the cells has nothing to do with your extracellular protein in that case.

    However, if you are planning to measure the expression of a RECEPTOR on the cellular surface for that extracellular protein, then I would suggest not to use trypsin because it is a bit harsh on the cells and may strip off your receptor, better use EDTA in that case. You also need to bear in mind the sub-cellular localization of this "receptor" if you envisage to do a Western blot using whole cell lysates, as the "receptor protein" may be localized in other sub-cellular compartments, not only on the cell surface. So you may need to prepare subcellular fractionation before you do your Western blotting.

  • Jörg Schüpbach added an answer:
    5
    Why am I not getting any result in WB from the primary hepatocyte culture lysate?

    From the primary hepatocyte culture of C. batrachus, I am getting protein concentration in the range of 7-10 µg/µl in Bradford assay. However, in western blotting, I use to load 50-75 µg total protein in each lane. I have used a range of primary antibody concentration from 1:2000 to 1:10000 but, still was not been able to get any band for mostly abundant proteins. In case of endogenous control also (GAPDH), a very faint band is coming. So, what might be the reason behind this or what should I follow to get result in WB. Moreover, what is the usual concentration of protein one gets from a primary culture? 

    Jörg Schüpbach

    Dear Debaprasad, you write "Moreover, in case of tissue, I use to get very convincing band with the same amount of total protein." Does that mean that your problem arises when using something else? Please identify the kind of sample you use, otherwise you cannot expect to get a useful answer.

  • Pavel Prosselkov added an answer:
    3
    Phospho WB issue: will tissue kept on ice before homogenization loose protein phosphorylation status or not?

    Hi all, 

    I need to detect the phosphorylation status of a brain protein. We use SynPER reagent for preparing synaptosomes from frozen brain samples and include phosphatase and protease inhibitors. Before homogenezation the tissue is thawed on ice and, dependent on the number of samples, may stay on ice for a while (up to 20-30 min). Do you think that this will affect the phosphorylation status of the protein i.e. is phosphorylation that sensitive? In addition, any advice on preparing tissue for phospho-WBs would be much appreciated.  

    Best wishes, 

    Tau 

    Pavel Prosselkov

    The kinetics of dephosphorylation depends on the target protein. If you are talking about neuronal activity markers such as CREB or MAPK there are losing their P marker within seconds even on ice and there is no magic protease inhibitors mix to help you, whatever the vendor says. If you do a time-course always shuffle your samples loading order, boil your sample right after -80C while it is still frozen (or better right after dissection) and add your denaturation buffer directly without thawing the sample.

  • Wai Kit Cheng added an answer:
    4
    What happens on my western blot membrane?

    Refer to the picture attached, it shows some of the lanes doesnt show a nice band. May I know any possible reasons or causes that make this happen?

    Wai Kit Cheng
    Hi Alexander, figure B is a different gel.. but I used the same receipt for both gels and run them at the same time and same voltage.. the antibodies I used for both of them were same
  • Priyamvada Pitale added an answer:
    4
    How do i prepare the positive control from a purified peptide for western blot?

    how do i prepare the positive control from a purified peptide for western blot?

    Priyamvada Pitale
    Thank you everyone. This is really helpful.
  • Rachel Bell added an answer:
    4
    What are your favourite western blot proliferation markers?

    I have some cells that I know are proliferating less after drug treatment however are not showing a decrease in PCNA by Western Blot, I have read in a number of places now that PCNA can also be expressed by resting cells and may not be the best option in my case. Ki67 is not really an option due to it's large size but what do you use as a proliferation marker by WB? Thanks in advance!

    Rachel Bell
    Perfect thank you everyone! I have some of those antibodies so will give Cyclin D1 and p21 a go.

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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