- Erik Maronde added an answer:Can anyone help with PKA substrate in HUVEC?
I stimulated HUVECs with Isoproterenol and expect that this would lead to increase in interacellular cAMP levels and hence activation PKA. I would like to investigate the activation of PKA by western blot. Does anybody have any experience or suggestion for any possible substrate for PKA present in HUVEC which can be monitored by western blot? Is CREB a good candidate to be followed?
Thanks in advance.
CREB would not be my first choice, but if you just want to know about PKA-dependent phosphorylation, try the CS anti-PKA-Substrate antiserum, which detects the PKA (PKG/PKC) consensus phosphorylation sequence.
Good luck, ErikFollowing
- Amy Schutz Geschwender added an answer:If I use more than two antibodies in the same solution for western blotting, what is the possibility for blotting on pvdf membrane?
If I use more then two antibody at a time for Western blotting but all targeted proteins molecular weight not same as others.
Yes, you can do this. The easiest way is two-color fluorescence detection, but that's not required. You should choose your antibodies (both primary and secondary) very carefully to avoid cross-reactivity. It's very important to run controls, to make sure that no cross-reactivity occurs between the various primaries and secondaries. Spurious background bands and/or cross-reactive binding could really interfere with interpretation of your results.
If you plan to quantify the Western data, controls become even more critical. Avoid stripping and reprobing if you want to do quantification - blotted proteins are often lost from the membrane during stripping.Following
- Kritika Sudan added an answer:Can anyone recommend a good source for purchasing NrF2 antibody (for western-blot analysis) ?I have some problem for Nrf2 detection with total proteins and nuclear proteins extracts
Nrf2 from Genetex. Molecular weight is approximately 92kDa.Following
- Mana Mahapatra added an answer:Can I use Triton X 100 in place of tween 20 during western blotting? If yes then what percentage should I use?
Tween 20 is widely used during western blotting. I wanted to know if I can use Triton x 100 in place of tween 20.
Cool- good luck with that.Following
- Ma Lingyun added an answer:Any advice on the time of transfer by semi-dry BIO-RAD for a 250kda protein?
hi,everybody.I'm just starting to do western blot,and so many problems came out.
I just tried to transfer 250kda protein,but failed all the time.So I need some advices and to improve my protocol.
I used 5% separating gel,and 1x transfer buffer (20% methanol),7v for 4 hours.After 4 hours,I found the gel transparent，and PVDF membrane do have the marker ,but fliter paper were yellow,I have no idea why filter paper were yellow.
Waiting for your help,thx so much
Thank you all. You guys help me a lot ,and now I can transfer the membranes well!!! Thank you guys so much~~~~~~Following
- Gopal Tiwari added an answer:Do I need to purify the protein expressed in P. Pastoris before deglycosylation with PNGase F?
I cloned my gene of interest in Picz alpha A vector and overexpressed it in P.pastoris X33. When I run SDS PAGE or do western blot I get bigger size protein band than expected which might be due to glycosylation of protein. So I wanted to ask whether I can directly use supernatant (having my expressed protein in it) for deglycosylation or do i need to purify my protein before deglycosyltaion?? And also can I use deglycosylated protein for western blot analysis?? Thanks
Sure Tom, I will try doing that as you suggested .Thanks once again.Following
- Dominique Liger added an answer:What 2x Laemmli Sample Buffer recipe is better?
Up till now, there are two kinds of 2x Laemmli sample buffers:
Buffer 1) 65.8 mM Tris-HCl, pH 6.8, 2.1% SDS, 26.3% (w/v) glycerol, 0.01% bromophenol blue
Please refer to the original paper: Cleavage of structural proteins during the assembly of the head of bacteriophage T4
And BioRad, Cat# 161-0737.
Buffer 2) 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromphenol blue and 0.125 M Tris HCl, pH approx. 6.8
Please refer to Sigma, Cat# S3401. And Cold Spring Harbor Protocols.
Buffer 1) is for optimal band resolution based on BioRad manual.
Does anybody know the advantage of buffer 2)?
The best recipe is the one which is working the best for your experiment!Following
- Daniele Paolo Romancino added an answer:I want to know why we use methanol for activation of PVDF membrane before western transfer?Why do we use nitrocellulose membrane without activation?
Methanol seems in order to displace the air trapped in the hydrophobic pockets of pvdf membrane allowing a subsequent replace of methanol with the water contained in the used buffer. See this link: http://www.pall.com/main/oem-materials-and-devices/literature-library-details.page?id=27294Following
- Othman Alghamdi added an answer:Why is my western blot black and without bands?
I am currently doing western blot for p-Akt. Below is my protocol:
-- Blocking = RT, 2 hours with 5% BSA
-- Primary antibody = p-Akt (1:2000), overnight, RT
-- Secondary antibody = Anti rabbit with HRP (1:3000), 1 hour 30 minutes, RT
-- Exposure = LAS Image 500
Result = black blot with no bands
I have noticed some thing in your protocol that if you want to incubate with primary antibody overnight, you need to put it in the fridge not at room temperature!!. we usually incubate overnight if we see faint bands, is this why you incubate overnight?. Other than that, I agree with most of the comments mentioned in this dialogue.Following
- Daniel Hoffmann added an answer:Can anyone suggests a good method to reconcentrate proteins after isolation? Precipitation, Speedvac or Lyophilization?I need to reconcentrate proteins with a very low concentration which are dissolved in RIPA buffer to do a westernblot.
In this case I would suggest to do an SDS-PAGE from the precipitated samples to check whether its possible to separate them by size exclusion. In case you´re lucky your protein is the largest or the smallest in the fraction and you could ust filters with respective cut-offs as described here before. If this is not possible you probably have to purify your protein via chromatographic methods. Size exclusion or Ionic exchange are methods you can use without an affinity tag like 6 His or GST present. Ion exchange requires the knowledge of the pI of your protein. With size exclusion this is not the case. In both cases it will probably be necessary to concentrate the eluted fractions from the Chromatography again.Following
- Ana Bagues asked a question:Is there a protocol to separate nerve terminals in muscle tissue?
I am trying to quantify mu opioid receptors in muscle by western blot but I am not geting good membranes to quantify, they have too much background and the bands are a bit smeared, I have already tried blocking with BSA and milk and different concentrations of the antibody. I have used other antibodies with the same protocol and they have turned out pretty good. I homogenize in RIPA buffer with protease and phosphatase inhibitors and charge 50 micrograms, heat the sample with laemmli buffer and betamercaptoethanol (95º 10 min) and charge 50 micrograms of protein in 10%acrilamide gels. Is there any protocol to sepparate nerve terminals in muscle? I am wondering if this will help get clearer results as I won´t have so many proteins that might smear the results. Thank youFollowing
- Sumeet Manandhar added an answer:Any suggestions on why in Western blotting I can not get the bands of Lrp1?
Western blotting :In first i could get the lrp1 both bands but now its very difficult to get the bands. instead i get non specific bands .for cross check i have done the western blotting of old sample also i got the result. i know that i have some problem in my cell lysate preparation. What could be the mistake?
Thanks Alejandro Martin. Its not the cross contamination. Now i have got the results. But i have one new problem. I have distort bands. I dont know why. If you have any suggestion can u tell me. I have attached the pic of my results.Following
- Augustine Rajakumar added an answer:How do I perform western blot analysis of endothelial nitric oxide synthase (eNOS) in human placenta?
Does anybody know of a good protocol to extract proteins from placenta? I want to see the expression of eNOS (135kDa). The biggest problem is the amount of blood in this tissue.
Second of all, I had problems with the transfer (I did 400mA for 1hour, but it didn't work well), in CAPS buffer.
Does anyone have any experience about this type of tissue and this type of protein?
Please see the two references below.
1. Novak J., Rajakumar A., Miles TM., and Conrad KP. Nitric oxide synthase isoforms in the rat kidney during pregnancy. (2004) Jnl Soc Gyn Inves. Vol 11/5 pp280-288
2. Rajakumar. A, Ketah Doty, Ashi Daftary, Gail Harger and Kirk P. Conrad. Impaired Oxygen dependent Reduction of HIF-1a and -2a proteins in Preeclamptic Placentae. Placenta. 24, 199-208, 2003.
You can contact me if you need further clarification.Following
- Jan Voskuil added an answer:Why is His-tagged protein not visible in western blot when recombinant protein gets expressed in the same molecular weight every time?
My His-tagged recombinant protein (MW-50 kDa) is fully over-expressed in desired molecular weight on SDS-PAGE. Before purification i need to confirm it through western blot. After various attempt i am not getting any band. I took another His tagged protein as positive control, which appears all the time but other protein does not give any sign of band. Here protocol and sample preparation is very perfect because same protocol and preparation works for the other various His-tag protein. What should i do? Any valuable suggestion would be appreciated....
Thanks in advance.
Amit is correct. Never bet on one horse. You need both a good working His-specific antibody and in addition a good antibody specific to the protein of interest. They both should confirm each other by comparing control and over-expressed lysates.Following
- Pawel Kozielewicz added an answer:Is it okay to use NanoDrop Lite Spectrophotometer to determine UV range of conjugate and toxin?
Is it okay to use NanoDrop Lite Spectrophotometer to determine UV range of conjugate and toxin? Please comment if any other suitable method for conjugate detection
I believe histone H3 should be a good control for nuclear fraction of any cellFollowing
- Shyam Bandari added an answer:Any suggestions on the problems with the Western blot analysis on Myeloma patient serum derived exosomes?
I have Isolated exosomes from myeloma patient serum using Exoquick kit (SBI)..and I confirmed the size using Nano sight (50 to 200 nm)..Till this point all fine but I have an issues performing western blot on these samples, when I add WB loading buffer and heat the sample...sample is getting viscous (gluy)..so I was unable to proceed further ....does any one have suggestions to circumvent this problem ??
Thank you all.... I have diluted my sample and performed the western blot.. and the results look fine, I could see the protein of my interest.....once again Thank you allFollowing
- Bruna Rodrigues added an answer:Its possible to use the sample after western blotting?I wish to submit a sample to SDS-2D following western blotting. So I´d like to know if is possible to remove that sample used at botting for another experiment, like mass spectrometry.
só uma coisa, o coomassie precisa ser o R-250, ao invés do normal.Following
- Hediye Erdjument-Bromage added an answer:What post-translational modifications help proteins migrate faster on an SDS-PAGE?
My HA-tagged protein A appears to have two more bands when another FLAG-tagged protein B is co-transfected(as opposed to a single band when A is transfected alone). The two new bands run right below the original band and I assumed the original band is post-translationally modified. One of the two new bands is only a little less abundant than the original band, whereas the other band is significantly weak. I was just wondering what kind of post-translational modifications would let the protein migrate faster. Thank you.
I am also a bit late in this response.
If a protein is migrating to a smaller Mr (faster migration) it is usually due to the presence of a degradation product. In my experience, most of the PTM's tend to cause the protein to migrate to a higher apparent molecular weight ( it causes slower migration). If it is an N-terminal truncation, you can easily find out by transferring the mixture onto PVDF and subjecting each band to Edman degradation. If the truncation is at the C-terminus, peptide mapping of each species may help..Following
- Jiayee Wu added an answer:Can anyone help with a problem with Drp1 S637 antibody for western blotting?I have a problem using Drp1 S637 antibody from cell signaling for western blotting technique. Although it should be at around 70-80kD, I get a band at 120KD. Is this a splice version or a dimer?
I usually get another slowly migrating band near 100kDa, and I think it's the sumolyation of Drp1.
- Ranu Pal added an answer:Dark bands on edges of Western Blot and faint development of bands in the center of WB, even in the loading control?I am trying to replicate an experiment previously conducted, and observed that irrespective of the samples loaded, on developing the WB by chemiluminescent substrate, the bands on both edges of the blot appear darker than the bands in center. So on looking from left to right dark bands slowly fade and become lighter, before becoming dark again on reaching the right side of the WB. This is particularly troublesome since this is observed even in case of the loading control (GAPDH).
I agree with most of the answers. There are few suggestions on this topic.
1. If it is pre-cast gel, check the date of expiry.
2. After placing everything for sandwich, run a 15ml tube across the sandich before clamping to make sure there is no air-bubble trapped.
3. Place a container full of ice inside the transfer tank so the chamber does not get too hot.
4. Incubate the blots in a container that flat surface so the blots get covered with the buffer containing either primary and/or secondary antibody.
5. Do not let your blots dry completely during chemiluminescence procedure.
Hope it helps.Following
- Varun Chandrashekaran added an answer:Can anyone help with the trouble I have in getting protein from mouse liver tissue?
Hi, so I have been homogenizing mouse liver tissues that have been snap frozen by first grinding them with a mortar and pestle with liquid nitrogen. I then put some of the ground up powder in 500ul 2x Laemmli Buffer, which I then homogenize with a homogenizer. After that I add beta-mercaptoethanol to the samples, vortex them and boil at 100 degrees for 10 minutes. I then let the samples cool down and then spin them at 13,000g for 10 minutes. The idea after that is to collect the supernatant and use that for Western blots however for my most recent samples I don't get very much supernatant, and a large amount of pellet / debri. And the supernatant I have used and collected for Western blotting hasn't worked due to no protein. I have used this protocol for heart samples which has worked fine. I tend to get much more liver sample than heart sample when grinding.
Therefore does anybody know how I can resolve this issue and get better homogenized mouse liver samples. I keep all pellets and debri just in case I need to go back to them to repeat certain steps, etc.
Many thanks for your help,
Use about 500-700uL of RIPA with PI and Phosphatase Inhibitor for 30mg of tissue. Homogenise samples gently to avoid frothing. After homogenisation centrifuge samples at maximum rpm for 15 mins at 4c. Carefully collect supernatant for further processing avoiding the fat layer on the top which can interfere with your experiments.Following
- Kyoung-Ho Pyo added an answer:Any suggestion on why different predicted band sizes appear for the same protein?
Hello all :)
I am choosing the primary antibody for WB analysis.
During look around, I found there is different predicted band size for this protein.
In same company, two primary antibodies are on the market.
However, the one showed the band around 40 kDa, while the other showed the band around 70 kDa.
Clonality (monoclonal), origin (rabbit) and tested cell line is same.
The only different thing is immunogen: full-length vs. 1-300 amino acid.
What is the reason for this different predicted band size?
and Which band is my targeted protein?
some times fusion protein is able to find in cancer cell lines as EML4-ALK, ROS-ALK etc. The fusion proteins are also considerable. There is another, lysis buffer elements are important. Co-IP buffer is able to keep protein-protein interactions.Following
- Dawn Fischer added an answer:Have you ever had any problem testing actin in adipose derived stem cells samples by western blot?
I have tried several times to detect the actin (by Santa Cruz Biotechnology) after total proteins extraction from adipose derived stem cells (ASCs), but, every time, I obtained the burning of the film. I don't know why. Does you have any advice?
I had a similar problem with patient tumor lysates. The actin I had been using from Cell Signaling (always with great results) was giving me a hit and miss signal. Some patient lysates just wouldn't show up. I tried an actin ab from Santa Cruz and then I had a full signal for the very same lysates. I'm assuming there were just differential binding. Try another antibody clone.Following
- TMP Raju Raju added an answer:Does anyone have set hands in purifying the his tagged recom protein (secretory) NGF?
I'm working on Novel protein therapeutics for peripheral nerve regeneration. My protein NGF (Nerve Growth Factor) was expressed in Sf9 cells and secretory protein was unable to purify under native or denature conditions. I have performed Ni-NTA agarose resin and followed by silver stain and western blot. Other side I also tried to purify using Q-Sepharose ion exchange chromatography followed by Ni-NTA agarose, but no change in purity.
Please advise if any one has done earlier
T M P RAJU
Firstly thank you all for your answers.
I have developed 4 novel chimeric fusion protein combinations and out of all Chimera 1 (smallest) has been successfully expressed in Sf9 insect cell system and purified to.
Yes, NGF and Chimera 1 has been successfully expressed as I have tested 6.5 microlitres of DIRECT media on western blot and clear band observed, but other chimeras aren't.
All buffers are at Ph 8.0. But now I have optimized purification using Talon (Cobalt, Ph 7.4) which is highly specific I found
But problem with rest of the chimeras which are expressed but not secreted into the media. I'm planning to purify these 3 chimeras from cells. I'm not sure How much I will gain from it
I welcome you all again for your valuable advise
I'm happy to discuss more in detail if any one of your in to similar field of work
- Steven Larsen added an answer:Any suggestions on the problem of getting band on coomassie staining for co-IP protein?
I am doing coomassie staining after pull down with the protein of my interest in order to do mass spec analysis. For it, I isolated different (A and B) subcellular fractionation, got an intense band on coomassie staining gel from A compartments and had good mass spec results from the intense band.
However, I could not see any band after coomassie from B compartments.
But in case of western I usually get very strong band on A and B compartments extracts using same antibody.
I used different protocols and modified some methodology (using dynabead protein A, surface-activated dynabead etc). Here I can't change antibody for some reasons.
The antibody I used and also I should use is just rabbit polyclonal antibody not tagging one.
Can anybody help?
As there are huge differences in sensitivity between immunoblotting and comassie staining and you are using purified proteins, it could just be that there is too much protein to compare instensities by western blot i.e the signals are saturated. you could try several dilutions of the samples for the WB analysis.
- Bilge Ercan added an answer:How can I solve my detection problem for a His-tagged membrane protein ?
I am trying to purify membrane complex in E.coli and elucidate the interaction between sub-units. I tagged one of the protein in the complex with His-tag and did pull down to see other subunits would interact with it. I was able to pull down other proteins in the complex but cannot see my His-tagged protein. Neither on SDS-PAGE gel nor on Western Blot. Does anyone see this nondetectable membrane protein before? Thanks for your help!
I have found the solution! Apparently, the His-tagged protein was eluting as well but since I boiled the samples in SDS 2x reducing buffer before loading to protein gels it aggregated! When I don't boil samples I can see the His-tagged protein and its interacting partners. Did you encounter with this kind of thing before?Following
- Célimène Galiger added an answer:Are you familiar with Western blot troubleshooting-non linear bands?
I face a problem with my western blots and I don´t know what the problem is. Actually, I get nonlinear bands. I have checked buffers, voltages, pH, gels and so on and I still get the same results. Proteins with high molecular weights are ok but from 50 kDa and lower, the bands are not linear. I enclosed one blot so that you can see what I am talking about. Thanks
Thank you all for your answers.
Actually I load 30 µg of proteins in each well and I will also increase the volume of the running buffer. Do you think that it can be a problem of protein denaturation?Following
- Jit Kong Cheong added an answer:Why am I not able to detect the LC3 band in western blotting?I am stuck with this for the past 1 year and would appreciate any help possible. I am trying to detect autophagy levels in my cells and I am unable to consistently produce the LC3 bands (most of the time there is no bands except once). The conditions I used are as follow:
Sample: A5-RT3 skin carcinoma cells lysed in Triton lysis buffer
Gel Running: 12% gel, 30ug protein/well, tried running both half and full gels (was told that running gel too far would make smaller proteins defuse)
Wet Transfer: Tried 70V 20 mins and 45V 20-30mins, Tried PVDF membrane and NC (.45um) membrane
Western Blotting: As we are using the infrared detection system, blocking is done with the commercial blocker, washing with TBST, LC3 Ab is from MBL mouse monoclonal.
Can someone tell me why am I not getting the bands? Is it due to the MBL antibody, the .45um PVDF/NC, the transfer condition, the amount of protein loaded (assuming that infrared is more sensitive and the amount of protein used is sufficient) or the infrared system that is the problem?Following
- Martí Cabanes Creus added an answer:Has anyone experienced problems when boiling neuron derived samples for Western Blotting?
When I boil my neuron lysates (grinded) with SDS-reducing buffer I usually get two different phases, a liquid one and a "thick-floating" one which I used to ignore until I discovered that my proteins of interest might be stacked there. Has anyone had this problem? If so, do you have any solution to solubilize the thick phase? I guess it may be partly lipid partly insoluble protein, but I am not sure.
Thanks in advance
Thanks for your kind answers. The thing is that I'm working with ipsc derived neuron culture cells (24 well plate), trying to study AAV infection, so my target proteins are the capsid proteins, which I know they are replicating thanks to previous studies I did.
I used to ignore that insouble floating material but on the last western attempt I did manage to load it into the SDS-PAGE gel, and even though it got stacked at the beggining, I could detect signal there once revealing the membrane, which made me think that the capsids may be actually there.
In fact, in this last western blot I even ignored the cells and I took only the media to go to the western, but the insoluble floating material appeared again after denaturation/reduction, so my guess it was that it may be dead cells causing that...but I'm not sure.Following
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.