Varun Chandrashekaran added an answer:Why does my western blot show a dark black blob ?
I was running a western blot to detect EphB4 protein in my cell lysates,I incubated with Human EphB4 Ab overnight and blotted it with Anti mouse secondary,I am seeing a dark thick rectangle around inof my loading lanes.the membrane was slightly bigger than the box in which it was kept for blotting the antibody.
Can this be the reason for the black blob on my blot ?
Are you incubating your antibodies on a shaker. If not your primary may be concentrating at a particular area on the membrane especially if the surface is not even.Following
Lakshmikanthan P added an answer:Can anyone suggest how I can overcome the faint bands in western blotting?
I am using DAB staining to detect signal for protein expression, but now a days I am getting faint bands? Even increased the protein concentration, fresh antibody dilution, reduced blocking steps etc., can anyone suggest be how I can overcome this?
Shashikiran G Bhat added an answer:Why is my protein getting precipitated, even at low concentrations?I am purifying a recombinant protein (host Rosetta) with his tag. I am getting reasonably good yield (4-5 mg/liter of culture). However after affinity purification using Ni-NTA column, during concentration protein is precipitating. I am using Amicon 10 KDCO centricons for concentration and I could observe that even at 0.4mg/ml concentration protein is getting precipitated. Protein buffer is 50 mM tris pH 7.5, 500mM NaCl, 5% glycerol and 10mM beta ME and pI is ~6.0. Protein is eluting at 250mM Imidazole, is imidazole causing the precipitation?
imidazole concentration was removed after affinity and that worked fine but after gel filtration (20mM tris-Hcl and 150mM Nacl) its precipitating in corning concentrator. Need help.Following
Are there any techniques, other than ELISA, for measuring secreted protein (21KDa) from the conditioned media?
Since ELISA kits are very expensive, was wondering if I could measure that with western blot technique? and If yes, how to measure the protein concentration? For the cell lysate I use protease inhibitor cocktail in RIPA buffer to extract the protein and measure it with bradford assay. But for the proteins in conditioned media (DMEM+10%FBS), I dont have any protocols yet. Looking forward to your help. Thank you.
If you use Western blotting, make sure that your samples and controls are within the linear range of your detection method. Film is not recommended (the linear range is impossibly narrow) but digital systems can perform well. Here's a link to a recent paper that looks carefully at the linear range of WB detection:Following
Geoff Margison added an answer:Can anyone help me figure out why my wet transfer is always incomplete?
I have been trying for months to get complete transfer from gel to membrane with no luck. I have no idea what to do from here. Please help. Here are my parameters:
My protein of interest is 18kd and 24kd
I use a 12% gel (I've also done a 4%-20% gel) [pre-cast]
I use towbins buffer for transfer
My running buffer has tris, glycine and sds (1%) pH 8.3
I do my west transfer covered in ice to prevent overheating.
I use a 0.2uM nitrocellulose membrane
I usually load 30-40ug of protein
I've tried 30V for 1hr, 45V for 1hr, 45V 1.5hr, 40V for 2hrs, 70V for 2hrs (V always constant)
I use the X-cell blot wet transfer module from Invitrogen
Now the reason I say I don't get complete transfer is because there is so much protein left on my gel after staining. I also stain my membrane and see very faint bands as well. My rainbow marker always transfers perfectly which leads me to believe the transfer worked but never actually does. Please help me solve this dilemma.
I see the same thing (our favourite protein is 24kDa!!), but usually, the lower molecular weight bands are transferred more efficiently than the higher m.wt. bands when you stain the gel post transfer. I have tried very high voltage (the transfer buffer was too hot to touch at the end) and that didn't help. I was also concerned about the proteins overloading the membrane (PVDF is better than nitrocellulose in my hands) so in a different experiment, I put a second membrane behind the first, and indeed there was protein present on the second membrane. My guess is that if you manage to electrotransfer all of the protein out of the gel, you will lose some through the membrane.
So Alejandro's point is well made: why do you want to transfer totally?
If you are thinking of quantitative westerns (which must include a standard curve of the same protein as you are trying to quantify, then you can assume at lease the same (in)efficiency of transfer), we tried that decades ago and gave up in favour of ELISA. At the time, our reading was that the membranes themselves were too inconsistent in their binding capacities.Following
Vidyadhara D J added an answer:Can someone give advice about the western blot transfer?
I'm recently doing a western blot about a protein which has both precursor and mature structure. And the molecular weight of precursor is 120 and 70 for mature form. I have conducted the transfer before and it goes well. But today when I repeat the western blot, I found that the transfer step was strange. After transfer under 15V, the marker stays on the gel while the blue dye penetrate through membrane into filter paper. In the past succeeded cases, the marker is supposed to be transferred onto the membrane while the blue dye(the band) stays on the gel. What's wrong. I reviewed my procedure and cannot found what's different from before.
I appreciate any respond.
For ur 120kD protein, to get consistently reproducible transfer, it's better to follow wet transfer rather than semi-dry...Following
Niharika Gaur added an answer:Transferring high molecular weight proteins in western blots?We Are trying to obtain efficient and reproducible western transfer of a 340 kDa protein. We routinely do westerns for proteins of 140 kDa and below with no problems. Does anybody out there have any suggestions for our 340 kDa protein?
can you please tell me what membrane did you use ?
Vivica Grotelueschen added an answer:How can I look at 3 kDa protein with Western blot?
I am trying to verify if ghrelin is present in cortical collecting duct cell lines. Ghrelin is approximately 3 kDa and difficult to find on western blot. Does anyone have any suggestions? Thanks!
I agree to the others, try Bis-Tris or Tris-Tricin gels. If possible I suggest you to buy them first (or ask for one in another institute/department :-)) to eliminate problems due to self-cast gels. If it works, you can think about protocols for making them yourself to save money.Following
Adam B Shapiro added an answer:What is the best option among the following for long term storage of protein sample for western blotting?
1. Store the extracted protein in a specific buffer with protease inhibitors at -20 degree. When you want to run the gel, thaw the samples, add 1x laemmli buffer+ heat at 95 degree for 10 mins and load.
2. Extracted protein in buffer + protease inhibitors + laemmli buffer+ heat at 95 degree for 10 minutes and then store at -20. Whenever you want to run the sample in gel, just take out, thaw and load.
3. Extracted protein in buffer + protease inhibitors + laemmli buffer without BME and store at -20. Just before running them in gel, add BME, heat for 10 mins at 95 degree and load..?
Which is the best method among these...? Please help.
I think #3 would work also. It just adds an extra step.Following
Pranav Joshi added an answer:Pellet is forming again and again in the supernatant after using HEPES lysis buffer for protein isolation! Why it is so?
I once tried HEPES lysis buffer for tissue lysis to obtain protein. After homogenization and sonication , i centrifused and collected the supernatant for protein as per the protocol. but later on again pellet was formed. why is this pellet again and again in supernatant too? . In order to confirm wht is this pellet . I ran a gel for B-ACTIN consisting on supernatant , that pellet solution and mixture of both in each well.. In that i got nice band for supernatant but a less intence band in the pellet solution .. so its clear that supernatant should be considered for for western . the main question is why is that pellet is forming again and again after centrifugation? .. Now i am using 8M urea buffer and all works fine.. is there any specific reasons for the use of urea buffer for western blotting ???? Is the use of urea buffer can hamper the quality of band in western blotting? Thank you in advance.
Thank you all of you for your valuable suggestions.Following
Yaryna Storozhuk added an answer:What are a suitable conditions for doing a western blot to look at p21 expression?
I've been having trouble doing Westerns looking at p21 expression. I've tried a couple of different antibodies so I think the problem is with the transfer/setup. When doing a Western blot to look at p21 expression is it better to use 0.22 or 0.45 um PVDF? Also, what transfer conditions are best for p21? 1 hour at 100 volts, O/N at 30 volts etc.
- Hi Kenneth, I usually use a standard RIPA buffer, it would detect proteins that are membrane or nuclear bound and its usually the most standard one to use
150 mM sodium chloride
1.0% NP-40 or Triton X-100
0.5% sodium deoxycholate
0.1% SDS (sodium dodecyl sulphate)
50 mM Tris, pH 8.0
Add protease/phosphatase inhibitor cocktail prior to using lysis bufferFollowing
Kay-Dietrich Wagner added an answer:How long can we use the protein lysate for western blot analysis of Histone deacetylases?
I have 2 month old lysate. I want to do western blot analysis for HDAc 1-7 using this lysate. Can I use this lysate? In other words I want to ask about protein stability of HDAcs in the lysate, whether HDACs remain stable for 2-3 months in the lysate or not? The cell line I am using for lysate are: H!299 : lung cancer cell and DU145 cells: prostate cancer cells.
If you add protease inhibitor when making the lysates, aliquot them and store them at -80°C, you can use the aliquots for years.Following
Ketil winther Pedersen added an answer:Having issues getting primary antibody to bind during Western blots involving exosomes.I have been successfully performing Western blots on cell lysates from human glioma tissue using tags for actin, HSP70, HSP90, and others, however, I cannot get the signal to show up when I include lanes with exosomes. Even when I alternate lanes (cell lysate, exosome lysate, etc.) I get nice crisp bands in the cell lanes but not in the exosome ones.
I have tried Laemmli 2x buffer as well as RIPA buffer. Transferring the proteins from gel to PVDF always works, for both cells and exosomes, and I get clear transfer in exosome lanes at the MW that corresponds to HSP70, which is what I'm most interested in.
give me a few minutes and I will put it down for you together with some more information based upon our experience and challenges.
Have a great weekend.Following
Mark K Chee added an answer:Can anyone help me resolve the background issues I've been having with Western blot?
Recently one of my blots came out looking like this,
I am trying to figure out whats causing this problem, the concentration of the antibody or something else.
My blotting protocol is
3x washes in TBS (with Tween 0.03%) after transfer
block o/n in 4%BSA
3x washes in TBS
1 hour primary at RT
3x washes in TBS
1 hour secondary at RT
3x washes in TBS
apply ECL 1 min
Could anybody tell me what's causing this
This blot was reprobed using 0.1M glycine (pH 2.5)
If you ever use PVDF/Immobilon again, here is an excellent stripping protocol that's much cleaner than the glycine method.
"A solution for stripping antibodies from polyvinylidene fluoride immunoblots for multiple reprobing"
Mark K Chee added an answer:What are the causes of a bad Western blot result?
I did the Ponceau on the last run, and it came out looking like these two images, attached.
What could cause this, besides bad gel?
Aleks, high salt concentrations will indeed cause aberrations on your gel (and subsequently the transfer). You might also notice precipitates forming after you boil; I have faced this same problem when I prepare lysates in high salt buffers, which I could not afford to dilute because of the need to preserve phosphorylated residues on my protein(s) of interest.Following
Inês Pita added an answer:Is anyone familiar with a protein concentration method for western blotting?
I'm using a new protocol for subcelular fractioning for rat striatum and frontal cortex. Unfortunately, I'm obtaining little protein in fractions (nuclear, sinaptosomal and non-sinaptosomal), especially in the striatum. I already used a pool of the cerebral regions but it's not enough.
Thanks everyone for the suggestions. I've been busy so I just tryed the precipitation method with ethanol, yet. However, I didn't get any protein again. I'm gonna try the TCA method now.Following
Daniel Perdiz added an answer:Does anyone have a specific protocol for checking DRP1 translocation from cytosol to mitochondria by western-blot ?
I have tried several protocols for mitochondria extraction but none of these were adapted for correctly visualizing by western-blot, translocation of Drp1 protein from cytosolic fraction to mitochondrial fraction.
In other words, has anyone an adapted mitochondrial extraction protocol adapted for this purpose ? Thank's!!!
Thank you Madhavika,
I think that the most critical point is the number of cells to extract.
Marco Bestagno added an answer:Which commercially available anti-His antibody has the best performance for immunoblot/Western analyses of 6His-tagged proteins?
I have tried a few primary antibodies to detect His-tagged proteins on westerns, and been disappointed with the low signal intensity compared to other primary antibodies used in my lab. I would greatly appreciate hearing about anti-His antibodies that you can recommend I purchase.
In our hands, the only one that gave consistent results was Clontech 631212. The only drawback is that it's extremely expensive.Following
Jesús V Jorrin-Novo added an answer:How easy is it to detect a difference in protein composition using proteomics alone?
A hypothetical question. I have two closely related plants. One is suspected to lack a certain protein present in the other. How difficult is it to discover this difference in the following situations:
A.) I know the exact amino acid sequence of the protein that should be lacking
B.) I have no idea which protein is lacking, I do not know its sequence
The task needs to be completed without the use of transcriptome or DNA analysis, only by the means of proteomics (Mass spectometry, Protein Electrophoresis, Western blot e.t.c)
Which methods should I use? How reliable are they? How long will it take?
In what case will this task be the most difficult to accomplish?
If you have got the sequence you shoukld make antibodies and assay by using dot blot first and then western.
have a look for the targeted, MS-based approaches, called "western MS" or SRM/MRM. If you have got a proteotypic peptide you may look for it.
For other general methods for comparative proteomics it is not possible to predict the results. As you have got the sequence, its Mr and pI can be predicted, so start with 1-D SDS PAGE electrophoresis and then move to 2-DE.
In any case we are not dealin with an easy or obvious experiment. It will take time and effort. Proteomics is not genomics.Following
Nirmalya Dasgupta added an answer:Use of Coomassie blue stain and ponceau stain as loading control in western blot?Is it reliable to use coomassie staining (before transfer) or ponceau staining (after transfer) as a representation of loading control?
As most people get problems in selecting a good loading control, why not use these staining methods to solve the problem?
I also faced the same problem for housekeeping gene in cell membrane fraction during colonocyte differentiation. I stained the PVDF membrane by Coomassie after developing the desired protein. You can try that.There is a beautiful paper on this, which I followed.
I got many new insights from several researcher through Researchgate, you can follow that thread regarding housekeeping genes. https://www.researchgate.net/post/What_are_proper_housekeeping_genes_in_the_plasma_membrane_fraction_for_western_blotting
All the best,Following
Emmanuel Aispuro added an answer:What about the size of a protein with 3 disulphide bonds in denaturing conditions? Could it be bigger than about 10 kDa in non-denaturing conditions?
I try to create a recombinant Kunitz domain (which has 3 disulphide bonds) in E. coli and in Pichia pastoris. The domain has a molecular mass approx. 13 kDa (together with His-tag). When I prepared a western blot and then I used his-tag antibody I have bands higher then I want. With E. coli it´s about 25 kDa and with Pichia 35 kDa. I use denaturing buffer. Is it possible to have these big bands after denaturation? Thanks
You could add up to 5 mM DTT to the the lysis buffer and so on in the next purification steps to avoid oxidation from the very beginning, once disulfide bridges are formed may be harder to breake them down and always a fraction remains as oligomers.Following
Mana Mahapatra added an answer:How do I raise an antibody from rat?
I need primary antibody (ACVR2A-Ab) for western blot. Help me how I can raising specific ab from lab animal?
You should have purified protein for immunization purposes - otherwise you may end up with antibodies mainly against cellular proteins. I believe you're looking for polyclonal antibodies?Following
Janice Ortega added an answer:Why could MSH2 be appearing in cytoplasm after nucleus-cytoplasm separation?
When I perform HeLa cell nucleus-cytoplasm seperation, I use human MSH2 as a marker protein for nucleus fraction. But the western blot show a sharp strong band in cytoplasm and nothing in nucleus. The antibody is bought from abcam (ab92473). Other marker proteins are all normal as predicted (HSP90 for cyto, HDAC1 for nuc). Could the antibody be mislabled? but the molecular weigh is correct.
Well then is very strange If is not your protocol. Because I have been doing nuclear extract for in vitro MMR and I never lost MSH2 into the cytosollic fraction.Following
Aleksei Rozkov added an answer:I am looking for a protocol to degrade rabbit knee joint tissue to do a western blot looking for collagen I and III. Any suggestions?Looking for a stepwise protocol and buffer and lysis buffer suggestions.
What about classic protocols with pepsin-aided digestion in acidic conditions?Following
Natascha Fussi added an answer:Did anyone of you know of a good antibody for NBR1??
I am trying some knockdown experiments with NBR1, but I have always problems to detect the 107 kDa band. In general the antibody is very unspecific and I already tried two or three different ones.
Furthermore there always appear one or two smaller bands on the blot, but I think they are unspecific.
Did anyone have an idea to get a better western blot result?
or a better antibody??
hi mafalda cacciottolo-thank you for the information I will have a look on your paper.Following
Thomas Keil added an answer:What can be the reason for the trouble with Dopamine receptor extraction and subsequent western blot experiments?
I've been using the "Mem-PER™ Plus Membrane Protein Extraction Kit" (Lifetechnologies) to isolate the membrane-associated protein fraction and also all integral proteins from mouse tissue according to the manufacturers protocol,
After loading a certain amount of protein (with an loading dye containing SDS in a concentration of 5% related to the whole sample and DTT ) on the SDS PAGE, i was not able to seperate the proteins by electrophoresis (almost all proteins remain in the upper third section whereas the marker migrate properly).
I used a stacking gel (4% polycacrylamide ; pH 6,8) and seperating gel (7% ,10% respectively ; pH 8,8) with following settings:
100V for 20 min (stacking)
180V, 130V respectively for a certain period of time (separating)
Has anyone an idea what i can do to solve this problem? Or what can be the reason for this poor-migration behaviour?
Normally dopamine receptor can be detected at 40-60 kDa, my results shown a band at around 180-250 kDa, indicating that proteins were not (badly) seperated.
Thx a lot for your help
Thx you all for your helpful answers.
We are going to re-prepare all necessary solutions.
If it will not work in a proper way, we will first use a RIPA buffer and replace the extraction kit afterwards. All commercial-available Abs seems to work but i didnt validate the specifity yet (positive controlls following). i'll keep you informed!Following
Salah M. Azwai added an answer:Can the IgM J chain on SDS-PAGE be detected?
While characterising purified antibody on SDS PAGE I ended up with only one band of approximately 14kDa, similar to the J chain, though no heavy or light chains. Does anyone know if you have the J chain present without the main heavy and light ones? More specifically if the purification method used was actually meant to bind to the light chain?
I would refer you to a protocol which I have used you with camels, elephants and ostrich it proved to be good. Check my list of publications, Good luck.!!!Following
Why would my lysates not settle down in the wells when I load them for western blotting?
As soon as I try to load my samles into the wells, it floats to the top and diffuses away, it does not settle at the bottom of the wells as usual.
What kind of loading buffer are you using? Maybe you don't have enough glycerol.Following
How to remove only ECL without removing most of primary and secondary antibody on the membrane for later re-developing the signal?
I would like to remove only just ECL and leave most of primary and secondary antibody on the membrane, then store it in -20C for later re-developing the signal with new ECL, may be in days, weeks, or month.
Can it be done?
If yes, what is the method should I use to remove just ECL from membrane? Is it washing? What to use TBS or TBST or Distilled Water or others? and How long of the washing is sufficient?
Thank you very much
The ECL reaction will eventually "burn out". All the substrate will be used up, and no more photons of light will be generated. ECL can only be detected while the chemical reaction is producing photons, so no reaction = no signals.
However, the HRP conjugate has a limited lifetime and the enzyme loses its activity. Although the concentrated conjugate is pretty stable, it is much less stable in dilute solution. I would expect freeze-thaw to destroy the HRP activity on the membrane.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.