- Arvind Gupta added an answer:My fusion protein has a MW of about 46KD. The band appears at about 40 KD, in purified form and in the lysate too. Can any one tell me the reason?
I have cloned a gene in pgex-4t1 vector, which is gst tag. My protein is about 20KD. While band appear at about 40KD. I have confirmed the clone by sequencing and double digestion. And also confirmed the protein using the gst antibody.
Did you confirmed molecular weight by MALDI?Following
- Laura Kytovuori added an answer:What WB blocking agents are there which do NOT contain dry milk or BSA?
I have an antibody which could not be used with blocking agents such as dry milk or BSA. What would you recommend for blocking? Please help me, as soon as possible :)
And commercial ones are excluded...
Thanks, both of you! I will try with TBS + tween20. I owe you one :)Following
- Asa Wu added an answer:How do you determine the maximum voltage for gel electrophoresis and western blot of proteins?
I read many protocols and Q&A in ResearchGate. Many can run protein SDS-page electrophoresis at 200V constant using same set of equipments that I use (Bio-Rad Mini-PROTEAN® Tetra Cell Systems). What are the conditions that they use to run 200V?
I tried once 150V for about 1 hr by using fresh cold running buffer (stored at 4°C). When the protein sample was running for about half a hour, the chamber became very hot, even in the cold room, and the gel started melting.
Is there a method to determine the maximum voltage for gel electrophoresis as well as for western blot of proteins? Can I determine it by looking at current and resistance values on the power supply unit?
Thank Claudio, I did store the buffer at 4C about three hrs before, and even put cool pack in it.Following
- Irene Serrano added an answer:How do you do a western blot for ubiquitin?
I am interested in ubiquitin western blotting.
I would like to know, if I use ubiquitin monoclonal antibody, will it give only a single band corresponding to it or will it be able to recognize other bands (where ubiquitin is covalently attached to other proteins) and give a ubiquitin ladder?
Kindly provide me with your precious advice.
The P4D1 antibody from Santa Cruz Biotechnology has given me nice and clean results. It is better if you use PVDF instead of nitrocellulose and denature the membrane before blocking. Good luck!Following
- Hoda Atef Elkhenany added an answer:Measurement of protein density of western blot.Does anyone know any good free software for measurement of protein density of western blot? I have tried "ImageJ", but there is a problem: when I select band and go in Analyse-Gels-select first lane, there is a problem with selecting second lane because it requires exactly the same size, but I cannot make the same size square.
So, How you analyze the data that you got from ROI manger, Are you dividing the ARea by the mean or what ?!!Following
- Mst. Nahid Akter asked a question:Can I keep bacterial supernatant at 80°C for few days before I concentrate the extracellular proteins and perform SDS-PAGE?
- Laila Al-Alwan added an answer:Can Beta-actin be used as a housekeeping protein for Western if Alpha-actin is affected?
For my inflammatory disease model, alph-actin is affected. Does anyone know whether I can still use beta-actin as a loading control for my Western blots?
Beta-actin or GAPDH both can be used as house keeping genes in westerns
- Clara Ibel Chamorro added an answer:Is anyone familiar with Type I collagen detection in Western blots?Can anyone tell me one good buffer which works best to isolate type I collagen from murine skin samples to detect it through western blots?
I have homogenized skin tissue and lysed in NP40 (normal lysis buffer) and also boiled directly the skin homegenate in BSB, in both the cases i could not see Type I collagen bands. Please suggest.
Wich conditions do you use for the transfer of proteins in your western?Following
- Clara Ibel Chamorro added an answer:Does anyone know where I can find mouse monoclonal primary antibodies for collagens 1-5,7,9,10,11 that will react in rabbit?I have found a few for cols I,VI, and IX but having trouble with others. I really need I, III and X. Doing Western blots.
May I ask which conditions did you used in the transfer of proteins?, I am having troubles with the transfer of high molecular proteinsFollowing
- Patrick Paumard added an answer:Is there a way that I can measure mtROS in fixed tissue? Has anyone had any success looking at proteins (not antioxidant) that measure mtROS via WB?
I already tried looking at mtROS using mitosox in primary cells. I would like another dye that I can use on fixed tissue
ROS have a very short half time life, thus, to my knowledge, it is not possible to detect relevant presence of ROS after tissue fixation.Following
- Ralf Max Leonhardt added an answer:Why has my co-immunoprecipitation stopped working?
I've been doing some co-IP experiments, pulling down my first protein of 55kDA and blotting for my second, which is 120kDA. This has been working nicely using an Active Motif co-IP kit. I have used 100ug protein and ~2ug antibody which is within the range they suggest to use.
The last two times I have done this experiment it has not worked. Previously I had seen antibody heavy and light chains with the 120kDA band (or not, in my negative controls). Now I am seeing a distinct band at ~150kDA in all lanes, positive controls and negatives. I am also seeing what I think must be the antibody light chain and much much more faintly, a heavy chain.
I have used a secondary antibody and a protein A/G conjugated to alkaline phosphatase to visualize my proteins but regardless, I see the same thing. The only thing that varies is the strength of the antibody heavy and light chains, as expected.
What is this mysterious 150kDA band?! And any ideas why my experiment suddenly stopped working? The only difference I can think of between my experiments that worked and the ones that didn't are that a new antibody aliquot was used. However I did a western blot to test this worked and it did, so I know it binds my protein.
Any help would be greatly appreciated!
Thank you in advance.
Heating is not going to reduce your antibody, as it does not break covalent disulfide bonds. Rather, your reducing sample buffer may have gone bad. I, personally, prepare my reducing sample buffer freshly before every experiment from non-reducing sample buffer to which I add DTT from a frozen stock.
If your starting material is a cell lysate, it might be a good idea to load a lysate control on the gel, so that you have a positive control (you know your 120 kDa protein should be in that lane). If your antibody detects your protein in the lysate but not in the co-IP then something with your IP is wrong. However, if the antibody detects the protein neither in the lysate nor in the co-IP then your Western is probably not working.Following
- Samantha Le Sommer added an answer:What concentration of IL-4 is used to stimulate murine BMDM?
I've been doing some signalling experiments with murine bone marrow derived macrophages with LPS and I was hoping to repeat them using IL-4 as a stimulant. I normally use 100ng/ml for 0, 10, 30, 60, 90, 120 and 180 minutes then lyse cells in RIPA buffer and prepare protein lysate samples for Western Blot.
I'm hoping to basically repeat exactly the same with Il-4 however I have very little experience in IL-4 stimulation and was hoping you would be able to advise on appropriate concentrations? I was thinking 5ng/ml of IL-4 however I've seen some papers use up to 20ng/ml. I'm looking for a concentration that will give me a signal that is not high enough to activate pathways other then the IL-4 pathway.
Many thanks for your help!
Thanks everyone for all your helpful thoughts. I think I might just do a titration at first. I'm only looking at very early signalling events (so 5, 10 mins) at least for this experiment though I am hoping to look at M2 IL-4 phenotype of the cells cytokine wise later on. I didn't realise you needed to have IL-4 over several days I thought it would be the same as LPS and I could do a 12 - 24 hour activation to stain for cytokines via flow cytometry. Thanks for mentioning it Fiona or I would have got all the way down the line and not realised! =DFollowing
- Komal k kumar j added an answer:What are the optimal ways to stimulate CD4+ T cells to look at Akt phosphorylation?I am currently using total splenocytes stimulated with plate bound anti-CD3 and CD28 to look at pAkt T308 levels by western blot (I will be using purified CD4+ CD25- Teffs in near future).
I noticed that the signaling happens within a few minutes of stimulation, however I seem to not get high pAkt increase in response to the doses I am using (10 ug/ml of pb anti-CD3 and 2 ug/ml of pb anti-CD28). I am using 5-15 min time points in 1% FCS complete RPMI. Am I hitting the cells too strong or too weak? Am I missing the kinetics?
There seems to be a variety of ways to stimulate CD4+ T cells to look at Akt signaling - If anyone could provide any suggestion or input, that would be great.
Can anyone help me to find suitable stimulation conditions to stimulate pStat1, 4, 5, 3, pAKT, p38 in mouse spleenFollowing
- Narayana Kilarkaje added an answer:What is the reason for problems with big errors after standardization of Western Blot results?
I am trying to check effect of degradation of some protein in cells after treatment with compounds. I performed few trials and I did standardization/normalization to loading control and to samples with my control compound - and trends every time are the same, but values differ a lot between each repeats... How is this possible? Some differences in f.ex. washing conditions or film exposure time should not affect WB after comparing to my controls, right?(but I try to keep always the same conditions)
When I try to calculate the average and calculate errors, th errors are extremely high even when results are in the same trend... Any idea?
One other thing. You can cross check your WB results by doing dot blot. If the latter is also giving no significant results, then the WB results are correct!Following
- Syed Zahid Ali Shah added an answer:What is the best possible pathway to check for the confirmation of axonal degeneration caused by prion peptides?
I want to work on in vitro axonal degeneration caused by prion peptide 106-126.
I want to confirm the best possible pathway of degeneration via western blot analysis. Can anyone help please?
Thanks alot for such a nice answer...Following
- Chao Xiang Ng added an answer:Is anyone doing western blot for caspase 4?
I want to recognize activated caspase 4 in TG treated cells using a western blotting assay. But only large subunits (19KD) of caspase 4 was detected in treated cells but not in control cells. On the other hand procaspase( 43KD) and small subunits were not detected in either control and TG treated cells.
1) As you had mentioned the strongest reactivity is with the large subunit. So it is not surprising that you only see bend over there.
This is a problem with antibodies which binds to both active and pro form, they might not bind well with the other subunit, even the abcam website is too ashamed to post a reference.
2) What you can do if you really want to see those bends will be cut your membrane if you are not already doing so, so that the 3 different kd range are separated.
a) Increase concentration of secondary antibodies (might cause film to become dirty if blocking is not done well) [I won't encourage this]
b) increase concentration of luminescence solution
c) Or increase the exposure time. Try an hour then 24h, I did before 24h for caspase 3... antibody was horrible,Following
- William C Lester added an answer:How to avoid saturation of positive control protein bands in Western blotting?
I am wondering how one can avoid saturation of loading controls while detecting low abundance protein of interest. I usually load 15-20microgram/lane of nuclear fraction from tissue homogenate to detect my protein of interest but this gives saturation of loading control bands (TBP in my case). I would appreciate your suggestions to overcome this problem as I want to quantify and compare the bands for various samples.
Beta-tubulin is also an option. Maybe try to use multiple controls in this case such as a nuclear protein from your sample. Diluting the antibody would work as well just not not have to do it myself. The only other thing is to minimize exposure using the method you are. Typhoon is great to use if there an 2ndary Ab you can use and you have access to a machine. Best wishes in your experiment.Following
- M. Ricky Ramadhian added an answer:Hi, has anyone ever used angiotensin ii to stimulate vsmc in order to test AT-1 by western blot?
I used different times and doses of angiotensin ii to stimulate vessel smooth muscle cells and then to test AT-1 by western blot, but none of the results had any differences with control. I just didn't know the reason.
How about make animal model induction with Ang II not with cell culture?Following
- Karolina Nowicka-Bauer added an answer:In 1D Western blot, why do I have that smear effect and some bands are white in their centers?
I have already reduced the concentration of primary antibody and incubation time with ECL. It worked but my 3 last blots look like that again. Any suggestions?
photo in the attachment
Thanks a lot Jordan! Yes, I reuse my running and transfer buffer :) The first one is commercially availalbe and they say it can be used up to 10 times but I use maximum up to 5, otherwise my electrophoresis would last ages. And yes, I will reduce the dilution of my primary antibody as a first one. Thank you all for the answears!!!Following
- Jordan Robin Yaron added an answer:What can I do when the band for loading control coincides with my protein band in Western blots?
During my western blot my protein gives band nearly between 42- 52 KDa.
I want to use beta actin or beta tubulin as my loading control. But, unfortunately it also gives band in the same region. How should I perform my western?
Please Help me.
The simplest answer is to use a different loading control antibody. GAPDH or COX IV are both sufficiently separated. Alternatively, multicolor detection using fluorescence or IR (e.g., LI-COR imaging as Ajaz said) is an option.Following
- Stefania Rigacci added an answer:Cell lysis buffer for western blot, which is better?We want to detect a membrane protein of ~120kD. The student used Mannitol/sucrose buffer for cell lysis - we did not see the expected up-regulation after treatment. However, immunofluorescence with exactly same treatment showed a ~20% increase. Every steps in the western blot was checked and was fine. I am wondering that since it is a protein located on the cytosolic membrane, the lysate buffer might be too gentle? Should we use RIPA instead? What is the advantage and disadvantage of using RIPA versus mannitol/sucrose?
Usually I rapidly scrape cells in Laemmli Buffer, vortex and then boil. If you want to preserve phosphorylation it is better to place dishes on ice while scrapeing. Sometimes I've added phosphatases inhibitors to buffer, but this does not significantly affects results.
I've never used Laemmli buffer to homogenize tissues, because this will produce a lot of foam....Following
- Rituparna Chaudhuri added an answer:How to identify if higher band on western blot is a result of a post translational modification or not?
I fractionate my cells into cytoplasmic and nuclear and consistently get a higher band in the nuclear fraction as compared to cytoplasmic when probed with my antibody of interest. How do I identify if this is a post translational modification? and if yes, what can I do to verify?
When I run the whole cell extract I still see two bands. None of the splice variants reported have that high a molecular weight (my nuclear band is about 10kDa higher than the cytoplasmic band). I am wondering if it could be sumoylation as there are reports that sumoylation drives some proteins into nucleus and adds approx 8kDa to a protein? in that case, can I check it using immunoprecipitation then western blotting with Sumo antibody?Following
- Hoorieh Soleimanjahi added an answer:Which type of membrane (nitrocellulose or PVDF) is best suited for transfering larger >100kDa proteins (western blot)?
*I am a western blot beginner*
The predicted size of my target protein is >100kDa. I have tried to transfer the proteins using a nitrocellulose membrane, but noticed that the higher molecular weight proteins remained in the gel. I understand there are other factors involved (conc. SDS and methanol in transfer buffer, transfer time, etc.), but wondered if a particular type of membrane is better suited for transferring larger proteins. Thanks in advance!
Both of them work well, but I prefer PVDF. You should optimize transfer time duration.Following
- Tomas Groh added an answer:Why am I getting a nonspecific HIF-1alpha blot?
I am getting nonspecific bands in HIF-1alpha blot. Can any one suggest why and how I can improve this?
I am using the following conditions
Blocking: 5% non fat milk powder
Primary antibody mouse: 1:1000 in plan TBST Santa Cruz
Secondary Antibody: 1:4000 in plan TBSTFollowing
- Hoorieh Soleimanjahi added an answer:How long we can store SDS PAGE gel before Western Blotting and what will be the effects?
Is there is any possibility that my gel will not suffer any damage/alteration in blotting, if I store the gel before western blotting?
It is preferable that the transferring of protein bands exactly after the running of them to prevent diffusion of proteins specially in non purified proteins. Although, you can keep the gel up to 12 hours at 4◦ C.Following
- He Huang added an answer:Does anyone have experiences with Immuno-Precipitation of ApoB?
ApoB is large protein, ~550KDa. I searched the literature and most of the published articles brifely described the prosedure of IP. They added SDS in lysis buffer and lyzed the cells overnight to dissolve ApoB. I followed these key points but got poor IP of ApoB. After centrifugation of the cell lysis, the supernatant was cloudy and I found ApoB existed in the undissolved part in the supernatant. Dose anyone have an idea to increase the solubility of large protein in cell lysis? Dose ultrasonication work?
Thank Thomas! I will try again using your protocol. Thank you very much!Following
- Fazel Gorjipour added an answer:Can anybody recommend a good antibody for westerns of p110-alpha in human cells?
We have tried several commercially available antibodies to p110 alpha in NETN extracts from HeLa cells but they do not seem to be picking up a band of the expected size. Can anyone help? thanks
I usually buy antibodies from this supplier: www.biorbyt.com
it works very well. I have never had difficulty with it. You can check it out.Following
- Vogel Walter K. added an answer:Does anybody have experience with blocking reagents compatible for the LI-COR Odyssey?I am using the LI-COR Odyssey system for Western Blot detection. I have high background levels
Nitrocellulose has a much lower background (esp 700 channel) than even the very lowest background PVDF we have tested. Nitrocellulose lots can vary in background (all low compared to PVDF) and rather than lot qualify these for ourselves we found that simply purchase the LICOR nitrocellulose, who do this themselves, the most cost effective route. As for blocking buffer, the LICOR product is great but is indeed expensive and we are looking for something more cost effective. We will likely give Aquablock a try; thanks Amritlal for the recommendation.Following
- Ajit Kulkarni added an answer:What happens when the pH of sample loading buffer in a western blot is higher than 6.8?
I am doing western. I prepared sample buffer using .5 M TRIS which was diluted from 2.5 M TRIS buffer stock having ph 6.8, I did not check the ph of .5 M TRIS and made the sample buffer using this TRIS . later on , after making the samples , I checked the PH of that .5M TRIS, It was near about 8. will this affect my sample or further process as for this buffer recommended ph is 6.8? And in one protocol, it was written that after making sample buffer adjust ph to 6.8. How can it be maintained as sample buffer contain glycerol, so it will be difficult to check ph with ph meter and it cannot be checked with ph strip too as it is already colored. so how to check. plz do reply. thanks
Nice discussions. I agree with this.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.