Antibodies - Science topic

Dear community,

I want to perform an analysis of the risilin localization within the legs of myriapods and would like to visualize the localization of the resilin components in different taxa.

Do you have any recommandations for a commercial antibody (doesn't matter if its mono- or polyclonal))

Thank you very much.

Best,

Benjamin

I have two antibodies that are staining well in IHC but not in IF for tissue sections, when using the same concentration and antigen retrieval method for both. I also know the secondary works well.

I want to deplete (negative depletion) all T-cells and NK cells from human buffy coat PBMC using three antibodies: CD3-PE, CD16-PE and CD56-PE and anti-PE magnetic beads. Can I do that by mixing the antibodies and following the standard protocol? How to calculate how much antibodies and beads do I need? How to calculate the yield of total cells I would get in the flow-through? I'm assuming around 30-40%?

If anyone has ever had a similar problem I would really appreciate it if you shared your experience.

I am wondering what folks have tried for alternative methods of detecting hepatitis B core antigen/capsid. Dako used to make an antibody that was very effective in this and widely used in the field, but since their acquisition by Agilent it is no longer made and there are no validated substitutes. We have already spoken to Agilent and checked Biocompare, and while several similar-sounding ones seem to exist, they all lack references or any figures showing that they work for western blots. All publications using immunoassay detection of this protein still use the Dako antibody. Has anyone found another antibody that works, even if you're not publishing with it yet?

As title describes, we're trying to mutate an amino acid of antibody Fc to cysteine, followed by TCEP to further drug conjugation, one of my colleague had raise the concern of purified TCEP treated antibody might be affected by the tris that had been used to store the protein. Is it possible? and if yes, what might be the reason?

I am working on Exosomes that is derived from cells that are engineered to express scFv of Anti-HER2 antibody. I want to see its expression in TEM. Can I directly label the exosomes with the Protein L-GNP or do I need to use Anti-Anti HER2 antibody and secondary antibody then stain with Protein L GNP?

Can anyone suggest literature on Protein L-GNP immuno gold staining in TEM?

I'm sure that many of you will relate to that experience of having an antibody that is just of bad quality. It creates problems because of cross-reactivity or non specific interaction and it's just impossible to work with, but is what you have or there is no other option but to use it.

So you soldier on, and develop new tricks to work with a fundamentally problematic system.

At what point you say.. "ok, it's enough" and admit defeat?

what is the last card you play before folding?

I was wondering if anyone knows an antibody that can be used for staining microglia in zebrafish? I am familiar with 4c4 and L-plastin antibodies, but these are not perfect for me. Is anyone familair with, for example, a P2Y12 ab that works in zebrafish ?

Thanks!

Hi!

I´m currently trying to isolate Mitochondria from HCM cells. For my western blots I need an additional antibody against a mitochondrial protein which is bigger or smaller than 14/17 kDa.

I was thinking of a protein which is only present in mitochondria or in the matrix. Maybe transmembrane proteins (TOM or TIM) will work too, but I don't know if the membranes are still intact in my samples. Maybe proteins from the ß-oxidation? Does someone know which antibodies can be used to detect mitochondrial proteins in western blots? If possible only proteins/Abs that are located/synthesized in the mitochondria (matrix). Thanks a lot!

We received a limited amount of an antiserum antibody as a gift from another lab and are trying to avoid having to purify it. Our initial titration blot with blocking in 5% milk successfully detected the target protein, but encountered significant non-specific binding likely from high albumin-IgG in the serum.

Would using BSA for blocking decrease the non-specific binding, or could it exacerbate the issue due to additional albumin from the BSA? We have never performed westerns with unpurified antiserum antibodies before so any help or tips would be appreciated!

Edit: This is NOT a phospho-specific antibody

Aslamo alikom/ Greetings everyone,

I'm conducting a western blot experiment (8% SDS gel) and I wanna test 2 proteins, one is 95 KDa, and another is 35KDa. The antibodies I've for both are mouse Abs used at 1:1000 dilution and I use secondary HRP-conjugated at 1:2000 dilutions.

Ideally, I use to test them sequentially, but I'm wondering if it's possible to add the 2 antibodies to the incubation buffer (BSA/milk) and test for both in one go?

I would highly appreciate an answer for this. Also, if someone has done it before, I would appreciate the feedback/tips if any.

Thank you

I am working on heterologous sandwich lateral flow assay.

I conjugate one antibody with carboxyl-gold, then print the other antibody on the nitrocellulose membrane.So the antigen should bind to the first antibody, then immobilize by the second antibody.

Even the two antibodies do not bind to each other, I always get two positive bands at the test line and control line when there is no antigen.

Different blocking condition(BSA%) and running buffer was tried, but I still see false positive band.

Is there any explanation or solution to this problem?

Hello. TetraHis antibodies recognise 4xHis-tag in the protein. What if I have a long His-tag (e.g. 10xHis-tag) and I perform ELISA or Western Blot based on native PAGE? Is there a chance that two TetraHis antibodies will sometimes bind to the long His tag and I'll get a false signal increase? Or is the potential distance between the antibodies too small and therefore after the first molecule has bound, the second molecule won't be able to approach the His-Tag, and it will be always 1 binding per 1 protein?

I started to think about it because the manufacturers of precoated well plates indicate the amount of histidines in the tag of the target protein. Is it possible that they imply potential overbinding?

Thanks.

i am working with membrane protein and there is a his tag 4 amino acid above the c terminus (seq HHHHHHDLEY). anti his antibody does not recognise the his tag in western blotting. i was wondering if it is necessary for his tag to be on N or C terminus for western blotting and purification

We have constructed lentivirus transduced U251 cell lines to express (1) EGFRvIII (its amino acid sequence is MRPSGTAGAAFLALLAALCPASRALEEKKGNYVVTDHG..., which is identical to the sequence described in https://www.addgene.org/20737/)-mCherry or (2) EGFRvIII_G38C(MRPSGTAGAAFLALLAALCPASRALEEKKGNYVVTDHC...)-mCherry.

Both types of cells did not yield positive results by using antibodies against LEEKKGNYVVTDHC (https://www.novusbio.com/products/egfr-antibody-dh83_nbp2-50599af647).

And the (1) EGFRvIII-mCherry U251 cells were also tested negative using 2-step staining (https://www.emdmillipore.com/US/en/product/Anti-EGFRvIII-Antibody-clone-DH8.3,MM_NF-MABS1915-25UG + https://www.thermofisher.cn/cn/zh/antibody/product/Donkey-anti-Mouse-IgG-H-L-Highly-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A32766),

The (2) EGFRvIII_G38C-U251 cells have not been tested by 2-step staining.

We are curious about this question. Have you met problems when dealing with EGFRvIII?

Prior to the research carried out by the research group who published this journal, they assessed HDV RNA detection's diagnostic efficacy utilizing the HDV RNA detection technique and discovered that it had a good diagnostic yield. However, HDV serology's diagnostic effectiveness hasn't been thoroughly assessed and documented, thus, the need to consider the factors needed in developing a reliable serological test for HDV antibodies.

Based on the systematic review and meta-analysis of Zhenzhen Pan et al. (2023) on the efficacy of serological antibody detection tests on Hepatitis Delta Virus, they concluded that detection of IgM or IgG is a better choice in HDV diagnosis compared to total antibodies, since total antibody pooled consistently lower specificities. In line with this result, can this limitation be addressed now by more recent or standardized serological tests?

I've received antibodies diluted in P.B.S. instead of 5% BSA in 1X T.B.S.

Will this dilution work for IHC or I've to discard the dilution ?

The cross-match test is an in vitro test to determine the presence of anti-lymphocyte antibody to donor cell antigens (lymphocytotoxic antibody) in serum of an individual with preformed antibodies to donor cells. Examples are recipients for an organ transplant or a couple with a history of recurrent spontaneous abortions. The recipient serum is incubated with donor lymphocytes and the binding can be detected by flow cytometry analysis (with fluorescent conjugated reagent). If cytotoxic antibodies are present in maternal serum, they will combine with the surface antigens of donor lymphocytes; the amount of fluorescence on the cells (percentage of positive T or B cells), as measured by flow cytometry, is proportional to the amount of antibody (flow cytometry cross-match).

I want to conjugate my antibody with Dylight-NHS ester and corboxylated fluorescent PS beads. How do I check whether both Dylight-NHS ester and corboxylated fluorescent PS beads coupled with my antibody?

I would like to enhance the fluorescent signal of mRuby3 in brain slices. I have only found antibodies good for WB. Did anyone try them on tissue? Is there a good antibody for IHC?

i am trying to stain different proteins of interest in human paraffinized section.

my signal should be the vessel only, but regardless of the antibody I get circle shaped spots , I tried antigen retrieval with trypsin, and I tried different dilution of antibodies (1:100-1:1000) and blocking in 5% and 10% donkey serum

why I have those spots? how can I reduce them? I have the same issue at different wave lengths (regardless of secondary antibodies tag)

this is my protocol:

•Thickness of sections : 10µ

•Deparaffinization by heat 55degrees for 20 min then (xylene - xylene: ethanol - ethanol) 10min each*twice

•Hydration (ethanol 95% - 70% - 50% - tab water) 5min each*twice

•Antigen retrieval : citrate buffer 10min microwave then leave in buffer to cool

•Peroxide treatment: 3%H2O2 10min at room temp

•Blocking: 5% Donkey serum + 0.3% Triton-PBS 1 hr RT

•Antibodies:

•Primary in 1%BSA: VWF (1:200)

•Secondary 1:500 of Rabbit 594

Almost all the microbiology textbooks and relevant research articles mention that Hepatitis B core antigen is not released into the blood of the host. It rather interacts with other core antigen particles to assemble the capsid of the Dane particle. My question is then how the body produces antibodies against HbCAg?

I was looking for an antibody that can block the ligand binding domain of mouse EGFR. Can I use Cetuximab or is it exclusively human-specific?

I don't want to give the mouse peritonitis, but I don't want to lose the antibody with it sticking to the filter either. Thanks!

Activation of naive T cells generally requires antigen to be presented by dendritic cells in lymph nodes. Activation of naive B cells generally requires opsonized antigen to be displayed by follicular dendritic cells in lymph nodes.

Thus, it seems that dendritic cells and complement (innate immune system) need to recognize a pathogen before an adaptive immune response can be initiated. Is this always the case?

Hello everyone,

I'm quitte at a loss here. I'm using the DYKDDDDK Tag Monoclonal Antibody (FG4R) (Catalog # MA1-91878) from thermofisher and while it works beautifully when I blot against Flag or use it to pull-down Flag tagged proteins. I can't see any signal when I use the same antibody to blot for Flag after the IP. If I use an antibody against the protein itself and not the tag, I have a beautiful signal and the shift due to the tag is clearly visible. When I use the Flag antibody, I have no signal.

Did anyone experience that issue ?

Thank you in advance for your help :)

Wilhelm Vaysse--Zinkhöfer

I am planning an immunoprecipitation experiment using Mouse monoclonal [11C9] to Mannan Binding Lectin/MBL (ab26277) antibody to immunoprecipitate for MBL2 in human serum. There are many protocols online for immunoprecipitation utilising cell cultures, where the recommended amount of cells tends to be 10^6 to 10^7, however not so many that utilise human serum.

Reading online, the optimal total protein load of immunoprecipitation seems to be 1-5 mg/mL, with 0.1 mg/mL being the minimum recommended load. Considering that the normal range for total protein in human serum is 60-83g/L (average: 71.5 g/L), loading 5 mg of total protein for immunoprecipitation would mean I need 69.9 mL. Alternatively, loading the minimum (0.1 mg), I would need 1.395 mL of human serum based on my calculations.

I cannot afford to be using 1.395 mL of human serum in my experiment due to lack of sample volume. I was wondering if anyone can share the amount of human serum they've used for immunoprecipitation before, where it was successful. Thank you in advance.

Dear experts and colleagues,

I am facing the ghost test line as shown in the attached photos.

Problems are as follows:

1) The flow was slowed down or stopped at the test line. It could not pass the test line and had to flow beside the test line (the dot).

2) It took time for the test line getting wet.

3) The test line became white (ghost test line)

It seems that the test line became hydrophobic that prevents the flow and interferes with the reaction between the Antibody at test line with the Antigen+ conjugation complex.

I intend to block the membrane by BSA 1%, Tween 20 0.05% in PBS 1X. However, I wonder whether I should immerse the membrane in the blocking solution BEFORE or AFTER dropping the Antibody to the test line? As I understand, I should block before dotting the protein on the membrane but I have read somewhere else that we can do it after immobilizing the protein. Therefore, I would like to confirm it. Please help me. Also, If you have any experiences dealing with these problems, please share your solution if possible. Thank you so much!

I have been using primary unconjugated TH antibody followed by a fluorescence tagged secondary antibody for the noradrenergic neurons of my brain stem sections. Due to some changes in my protocol, I have an idea of using primary conjugated tyrosine hydroxylase (TH) antibody. Is it possible to conjugate TH antibody to fluorescence tag. If so, suggestions on available brands of them will be highly helpful. Thank you!

Dear all,

I am in search for ideas for what might cause the following issue:

I have done IHC-IF extensively and wanted to perform stainings on cultured cells now, too. However, I am observing unspecific staining of all primary antibodies tested. It is a mystery to me!

Here are the details:

I am staining murine cell lines that have been fixed with 4% PFA for 10 Minutes.

Protocol (washes performed, but omitted here):

Results: Three different cell lines (neuronal, astrocytic, endothelial) all stain equally for the antibodies used (broad variety from different species), e.g. anti-GFAP, anti-GFP, anti-MAP2... Most antibodies tested work well in IHC (some are untested) and - based on the literature - many of them are used in ICC successfully. A control without primary antibody does not show staining, so autofluorescence or unspecific binding of the secondary antibody do not seem to be the issue. The composition of the media the cells were cultured in differ between the lines.

Steps taken so far:

- Used new buffers, aliquots, cells etc.

- I have done a dilution series with two different antibodies:

I have also planned to do a qPCR next week to verify the identity of the cell lines just to be sure, but I am feeling like it is a technical problem. I cannot come up with a reason that makes the specimen "sticky" for different primary antibodies, but not for secondaries. It seems unlikely to me that there is a problem with all primary antibodies tested, especially since they work well in free-floating slices.

Any ideas what might cause it and how to solve it would be very much appreciated!

Henrike

Colleagues, good afternoon! We plan to conjugate antibodies with a fluorescent dyes. One source says that it is possible to conjugate anebodies that already have tag in the form of HRP, for example. Another source says this will be ineffective. How do you do this kind of work? Is the dilution of such a conjugate different from the conjugate with horseradish peroxidase?

We have the peptide and mRNA sequences; is it possible to create an antibody?

Hello all,

I've been struggling to get good FAP staining on my tissues using fluorescent IHC. I've tried three different antibodies from different companies, but the staining isn't working well. Has anyone used an anti-FAP antibody for human cancer tissues and gotten good results confirmed by a pathologist? Any advice would be helpful. Thanks!

We have mRNA sequence and peptide sequence if possible design antibody?

The old antibody has been discontinued and the currently offered are detecting a band at 62 kDa, which does not reflect the correct molecular weight (47 kDa).

My student optimizes the RNA dot-blot method using a biotinylated cDNA probe. The aim is to detect a positive hybridization signal of a probe complementary to transcripts of a housekeeping gene for Human actin beta in the human RNA samples. So far, we have not been able to detect consistent hybridization signals. Either there were non-specific, very weak or no hybridization signals at all. Above it, we have recently observed a reverse white signal in dot-blot spots with our human RNA after colourimetric detection. The only such phenomenon I found when googling for the solution appears after chemiluminescent signal detection. The alleged ghost bands or spots appear when a sample or antibody excess consumes chemiluminescent substrates too quickly. However, in the case of colourimetric detection, the chromogenic substrate accumulates instead of being consumed, even in the excess of both sample and antibody. In search of a solution, we performed a dot-blot with a matrix of sample and antibody amount combinations, which resulted in either reverse or no signal et all. Does anybody have any idea where the problem could be?

Please, see the attached photo of the dot-blot results. There are 3 membrane strips, the numbers 1, 3, and 5 at each strip indicate the antibody dilution 1:1000, 1:3000, and 1:5000 respectively. The pink strip on the top of the photo shows the identity (and in the case of RNA samples also the total amount) of loaded samples in the indicated order. There are total RNA samples at the concentrations 5 ug, 500 ng, and 50 ng, followed by positive control spots of biotinylated lectin (positive control of colourimetric development of a signal produced by reporter molecule HRP in conjugate with streptavidin) and the rightmost spot is the labelled probe.

I will be very grateful for any suggestions!

Hi all,

We are looking for an antibody that recognises N-terminally tagged His tag on a recombinant protein, any recommendations or where to find this information would be appreciated.

Thank you

I am developing a protocol for the generation of antibodies using the Phage display method. Could anyone share a protocol for bio panning of biotinylated antigens?

I would like to know if it is possible to use antibodies (in this case I would like to use two markers of neutrophils and monocytes such as FITC anti-mouse Ly-6G and FITC anti-mouse Ly-6C) whose application is referred only to cytometry for visualization in confocal microscopy.

Hello¸

Im PhD Student in UQTR ( Canada), I have a crucial experience for my project thesis : I have a WB (western blot) membrane that I need to store for a long time for incubation with other antibodies (Precisely Ubiquitin) . I read that I can put it in a bag at -20C.

I want just to confirm if I did it correctly (see picture) or there are other ways.

Thanks in advance

Ayoub

I want to specifically block a GPCR on cell membrane (also has mitochondrial distribution), but all commercially avaible drugs are cell permeable. therefore, I was seeking to use antibody to block the GPCR located at the cell surface. The antibody i used can identify the protein by surface staining during flow cytometry and also can pull down my target protein by immunoprocipitation, does that means this antibody can block the extracelluar region of this protein and be used for my purpose?

I am trying to detect CCK in plasma with the antibody. if anyone knows how to detect and how much to load.

The detailed Western Blot procedure

The membrane is first rinsed briefly with 20ml of TBS, followed by a 30-minute incubation, shaking with 10ml of Blocking Buffer. During this incubation, the volume of primary antibody needed for a 1:5000 dilution into 10ml of blocking buffer is calculated, and 10ml of blocking buffer is prepared for the antibody. Subsequently, the Blocking Buffer is removed, and the membrane is incubated for 45 minutes, shaking with 10ml of primary antibody in Blocking Buffer (Rabbit anti-ADH diluted 1:5000). After removing the antibody, the membrane is washed three times for 5 minutes each with 10ml of Blocking Buffer. Meanwhile, a 1:5000 dilution of the secondary antibody in 10ml of blocking buffer is prepared. The membrane is then incubated for 45 minutes, shaking with 10ml of secondary antibody in Blocking Buffer (Alkaline Phosphatase conjugated Goat anti Rabbit diluted 1:5000). Following the antibody removal, the membrane undergoes three 5-minute washes with 10ml of TBS/T. Finally, 5ml of BCIP/NBT liquid substrate (Sigma-B1911) is added, and incubation continues until color develops, with the reaction being stopped by rinsing with distilled water.

I was told I might have washed it with a different TBS (10mM one instead of 5mM)

What could the reason for such a different bands on the well.

I am hoping that someone can help me find an antibody for GABA, GAD 65 or GAD 67 that works well in marmoset cerebral cortex

Thanks

I have tried 4 IBA1 antibodies and cannot seem to get good staining. Mice were perfused/fixed with PBS/4%PFA. Brains were placed in PFA overnight, and then moved to 30% sucrose and frozen in OCT after sinking. My sections typically tend to be thicker (30um or 35um), but we do sections on slides instead of free-floating due to less handling and integrity of the structures. All of my antibodies work except for these IBA1 antibodies. I have tried permeabilizing with triton and saponin and got similar results. (Fix for 5 minutes on slide with 2% PFA, perm with 0.3% triton 15 min, block with 10% goat serum 30 minutes, then primary and secondary incubations with blocking serum). Antibodies are spun before addition.

Can anyone advise me as to why these IBA1 antibodies are creating so much background at both 1:100 and 1:1000, and why it is not staining the filaments of the microglia? Any advice is greatly appreciated.

Hello, 

I am using a c-myc-tagged plasmid for coIP experiments, and i always get two specific bands on my western blot when I immunoblot with the c-Myc antibody (ab32). I dont understand what can be the second band i see on my western blot. Can anyone Help plz 

Thanks 

Pam

I'm wondering if there are any cases in which a nonsense mutation don't trigger non-sense mediated decay. If so, I would like to know if it would be possible to detect truncated protein in a Western blot (if the antibody binds to a region that is present in the truncated product).

Thank you very much.

In our experience, the antibodies #9196L and #9192 from Cell Signaling have not been effective.

Agglutination of RBCs is normally caused by IgM antibodies. Is there any case in which IgG antibody caused the agglutination of RBCs?

Does anybody know how to convert IU/ml in case of anti-TSHR antibodies (Graves disease)? I would like to know how much anti-TSHR Ab I have in a sample

I am trying to create a workflow for flow cytometry experiment sample prep. I intend to use LIVE/DEAD™ Fixable Aqua Stain and an antibody cocktail for extracellular antigens.

I intend to use the viability dye first. The protocol I found does not seem to mind the protein content in buffers, as the cell washing is performed with PBS (5-10% fetal cell serum(I imagine they meant "calf")), and the cells are suspended in the same buffer before viability staining. I read that the presence of proteins in the buffers might contribute to a higher background, is that correct? How should I adjust the solution (for washing and resuspension) if that is correct?

Additionally, I intend to perform antibody titration for the antibodies I'm using. There are a few questions here too. We have no prior experience with these antibodies in the lab. Questions:

1. Is it important to do titration for every antibody in the cocktail?

2. Do I keep the same general sample prep with the antibody cocktail, only swapping the antibody cocktail with a single antibody that's in different concentrations? Do I also apply the viability dye as well?

3. The antibody cocktail I am to use includes dyes such as SB and BV etc, which require a special staining buffer or blocking buffer. Do I need these special staining buffers or blocking buffers for titration as well? Is it necessary since titration typically only uses one dye at a time (together with viability dye?)?

I know the information above might not be detailed enough but could you share your personal experience related to these scenarios? The protocol that I base my protocol on comes from this page

Thanks a lot for your input!

I am trying to understand the different phosphorylation states of SRSF1 protein in the cytoplasm vs nucleus and looking for specific antibodies for the phosphor state. As SR proteins include various SRSF proteins I do see anti-phospho SR antibody clone 1H4 which can detect all SR phospho states. I am wondering if it only detects hypo or hyper SR proteins and it can distinguish between the two states or else different phospho antibodies are available to study this.

Thanks in advance!

Hi

I am working on lair2 and it's interaction with C1q to understand who that affect lupus.

We used cell line to produce lair2 . than we transfected it with c1q.

and now we are doing western blot , to detect the two proteins, the first time we incubate them with c1q antibody . and we see the two proteins . after that we used the same membrane to detect lair2 , we wash the membrane and incubate it with lair 2 antibody and we see the protein .

we used 2 negative controls , in the first detection we saw two faint bands in them . in the second one we didn't see them.

I don't know what's the problem , and how to analyze it?

I have not found any anti-iGluSnFR antibodies available, so I am curious if standard anti-GFP antibodies could recognize some epitope in the circularly permuted form of GFP in glutmate sensor iGluSnFR... Does anyone have some experience with it?

Hello researchers,

I'm currently working on a project involving LC3B antibody (Cell Signaling & SIGMA) for western blot analysis. Unfortunately, I've been consistently facing issues with dark background and black patches on the membrane. Despite trying various troubleshooting methods, the problem persists. Has anyone encountered a similar issue and found a solution? Your insights and suggestions would be greatly appreciated.

Thank you!"

I want to know whether clinically any one is looking for anti HLA -C antibodies against spouse / Partner in Recurrent abortions? Which method do you find more appropriate Flow crossmatch or Luminex ?

Another etiology described is shared HLA alleles : I found very limited papers on the subject and request members of Researchgate to provide inputs pleasse.

I looked at the data sheet provided with the antibody, but I have found nothing regarding the recommended dilution range

Hi, I'm trying to isolate my protein (Flag-tagged NOD2) in native conformation for BN-PAGE. I plan to IP the NOD2, I need to get the NOD2 off the beads/antibody without denaturing it. If I add DTT (let's say between 100-500mM) will this separate the protein and the antibody? Since IgG is held together by disulfide bonds, I'm thinking that the separation of the heavy and light chains would disrupt the protein-antibody interaction.

Has anyone tried this? Do you have any tips for an alternate method to isolate my protein in its native conformation? Thanks!

Hello,

For many phosphorylation studies of MAP kinases, cells are serum-starved overnight prior to stimulation. Serum-free media such as DMEM 1X is supplemented with 0.5% bovine serum albumin. Is there any drawback to including NEAA in the serum-free media? Is NEAA known to cause phosphorylation of MAP kinases?

Thanks!

I'm doing this since the ordered antibodies were not received yet?

I am doing a ChIP-seq with a polyclonal antibody. In order to cut down on non-specific background, I am doing a pre-clear on my samples after sonication. Basically, I rotate the samples in a +4 fridge with unblocked Dynabeads for an hour or two, separate the samples from the beads, and add the antibody.

My question is whether or not I should take out my Input Control before or after the pre-clear. I've heard of labs doing either one. Since the Input Control is used to normalize your signal to the non-biased DNA, I feel like it could go either way. If I take it before the pre-clear I would be normalizing to the total unbiased DNA collected from my samples. However, if I take it after the pre-clear, then I'm normalizing to the unbiased DNA that my antibody is actually collecting the chromatin from. Does anyone have any advice here?

Thank you for your time,

Jennifer Cheng

Hello everybody,

I have a question related to western blot.

I'm studying the activation/phosphorylation of p65-NFKB in monocytes over time using three different stimuli. The transfer went well (as you can see from ponceau red), and the protein load is equal across all samples, however, two bands are partially missing. Could someone please explain how or why this might occur? Also I would very much appreciate suggestions on how to fix this issue and maybe avoid it in the future.

thank you all very much!

Ouis

Hello, I'm treating several immune cell lines with proteins tagged with His tag and want to test their binding to the cells using flow cytometry with an APC anti His antibody. However, I get a very high signal, as opposed to unstained cells, when I'm using the antibody on non-treated (but stimulated) cells. I would appreciate to hear if someone has an idea why anti His antibody stains regular cell lines that obviously are not suppose to express His tag on their surface ? Is it because the cells are stimulated? Thanks

Hi flow users/cell masters,

I'm just wondering if anyone here has experience with staining cytoplasmic cytokines after staining extracellular proteins. So basically what I did in my protocol was staining extracellular markers using PE-conjugated and APC/Cy7-conjugated antibodies, then fix/perm my samples using the 1X fix/perm buffer eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set before staining cytoplasmic cytokine using antibodies resuspended in 1X perm buffer.

I didn't see any APC/Cy7+ or PE+ populations in flow, which is weird coz the markers are supposed to be highly expressed. I saw some posts on Reddit that APC/Cy7 is not stable, but I don't know if PE is unstable either. Also, does anybody know if eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set contains methanol? I couldn't find the info anywhere, if it is, it makes sense then... Since PE and APC tandems are not methanol-resistant.

Thanks in advance to anyone who's going to answer this :)

I am searching for antibody to rat brain tissue. I would prefer to purchase antibody for both Western blot and Immunohistochemistry.

Thank you in advance.

General difference between antibodies and alloantibodies

Hello everyone. I am having some problems with an immunofluorescence experiment. My aim is to use the IF-CBA method for human samples and get negative or positive results.

Please check these reserch to learn about some details about IF-CBA（DOI:10.3389/fneur.2023.1289810 or DOI:10.21037/atm-21-3072；these two can read on resarchgate.）

My experimental system uses HEK293T with PCdna3.1 plasmid transfected with characteristic proteins (e.g. AQP4), then fixed (4% paraformaldehyde), washed and incubated with Triton 100x (0.3% Triton-100, 2h) and last closed (5%BSA, 1h). Subsequently, different experiments were performed. When I stained with the standardized antibody and the corresponding fluorescent antibody（both from abcam）, it showed good results; however, when I next incubated with human serum as the primary antibody, there was no significant fluorescence from either the FITC-labeled human IgG antibody or the cy3-labeled human IgG antibody. Why is this phenomenon occurring when the human original sample is known as a certain positive sample? Should I perform antigen repair? Thanks for reading.

I am currently in need of an antibody targeting gene X, specifically focusing on a distinct splice variant where the major variance lies within the N-terminal region. Unfortunately, suppliers like Cell Signaling and Abcam don't provide epitope information, which is crucial for ensuring specificity towards my variant of interest.

Does anyone have suggestions or experiences on how to acquire epitope information from antibodies offered by these suppliers? Alternatively, could anyone recommend reliable strategies or resources to verify the specificity of an antibody towards a particular N-terminal epitope of a specific splice variant?

Your insights or guidance on obtaining this essential information would greatly aid my research. Thank you in advance for your help!

I bought from Epigentek the EpiQuik DNMT Activity/Inhibition ELISA Easy Kit P-3139-48 and the CA capture antibody must be stored at 4°C but I put in at - 20 °C.

I bought from Epigentek the HDAC Activity/Inhibition Direct Assay kit P-4034-48 and the HO4 capture antibody must be stored at 4 °C but I put it at - 20 °C.

Does it happen also to someone? Is it possible to use it? Do I have to remove from freezer and put in fridge?

Thanks if someone can help me

immune system of human

Hi all,

I recently started working with flow cytometry with no prior knowledge. I have been trying to stain cells to practice flow cytometry techniques, such as panel setup, gating, and data analysis.

I have come to realize that the cells I use for practice don't particularly express the proteins that my marker antibodies selectively bind to. The sample prep itself is also tedious and not super efficient considering that I want to learn about the practical part of the instrument.

I want to use fluorescent beads for practice but am swamped with endless choices. Could you recommend two types of beads that are different in size and fluorescence labeling? If you have budget-friendly options, that'd be even better.

In case you know of other ways to easily practice flow cytometry operations, I'd be glad to hear them. Thanks in advance for your help.

can you provide me with the best p16/ki67 dual staining protocol for FFPE tissue (cervical)? and what are the best antibody brand for that?

How can I improve its stability?

For the western Blot, I need to check another antibody in my membrane but this antibody is from the same species as the first antibody so how can I do that without stripping? (Note: two antibodies are around 15 KDa away from each other.)

I am working with NOVATECH ELISA KIT for Brucella. In the leaflet, it is mentioned that it is a qualitative assay with a cutoff starting at 11. When I enrolled the assay through the DS2 automatic ELISA machine, the layout results showed values as numbers, not only positive and negative. What does it mean? Are those numbers the real titre value? Are they the real concentrations of measured antibodies? If yes, why has the company not highlighted the kit as a quantitative ELISA?

Hi, everyone!

I am a graduate student of pharmacology from China. I am trying to measure the plasma NETs level with anti-MPO antibody and Sytox Green, which are available in our lab. Here's how I did it.

Firstly, a high-binding 96-well plate were coated overnight at 4 ℃ with anti-MPO antibody(1 μg/mL, Thermo). The plate was washed 1 time with wash buffer, then blocked with 4% BSA in PBS supplemented with 0.05% Tween-20 for 1.5 hours at room temperature. The plate was washed 3 times again, then incubate with plasm (100 μL) for 2 hours at 37 ℃, 300 rpm. The plate was washed 5 times before incubating for 15 minutes with Sytox Green in dark (100 μL, 1:1000, Thermo). The fluorescence intensity (excitation at 485 nm and emission at 535 nm) was quantified.

But there was no difference in fluorescence intensity between plasma and negative controls. I'm not sure what went wrong. I hope anybody who did it can give me some advice. Thank you so much for your generous help!

Best wished!

Yafei, Fang

Hi All,

We routinely use HEK293 cells to produce recombinant proteins and antibodies in our facility and we observed that for a small portion of proteins/antibodies, there appear to be a non-protein type of aggregates that co-elute with the target protein/antibody during purification. Such aggregates can be visualized as precipitates that have a string-like appearance and can be removed by centrifugation. The removal of these aggregates doesn't alter the protein/antibody concentration and they will re-appear after 4C storage. I was wondering if anyone else might have faced a similar problem and I would appreciate any suggestions on how to remove or prevent such aggregates from forming.

P.S. We use a culture system similar to that of EXPi293.