I have a problem with not properly transferring proteins on Nitrocellulose membrane. The transfer protein look fuzzy and all over the membrane as you can see in the picture. The protein marker was transferred but something is wrong with protein running on gel or transfer or may be my sample is old which is whole cell or tissues lysate 4, 5 months old at -80 C.
Another thing I have noticed that my gel enlarged in size after transfer which is also something I haven't seen before. In the transfer buffer I add 20% methanol, don't know if it's something because of it.
I use pre-cast gel and immunoblot module of invitrogen, I use the setting they recommended. For gel run I use 125v at 125Amp 3 Watt for 90 min setting and for transfer 25V constant starting current 125Amp for 90 min.
I don't think your sample is old, I used tissue lysate which was in -20 after one year.I think after SDS-PAGE you stain the gel, see how it looks, it might be something wrong in first step. Make sure your running buffer and transfer buffer are not old.
Have you used this sample before? you check the protein quality and concentration?
That also tends to happen when the transfer buffer is old or heating. Us Fresh buffer and make sure the transfer buffer is not heating
I assume you are using a semi dry system, isn't it? I've seen a similar thing with the bio-rad semi dry system, when I transferred 4 blots at the same time...It was a matter of position on the plate where you assemble the sandwich (don't ask me why...). Anyway, I need more infos. Is it a reproducible behaviour? I mean, is it happening always? Did the gel run good (without smiles or other strange things)?
Thanks to all of your suggestions.
I don't know how to check quality of my proteins. This is a wet transfer and I am sure I positioned it correctly on the plates. All the buffer and reagents were freshly used, no smile shape at gel running. My protocol was working fine but now suddenly this thing happens for continuous 3 times. I don't know there shouldn't be heat generation as I check the temperature during transfer that is around 15-20 C. I am completely puzzled what could have been doing this.
Whenever I run WBs, I run 2 identical gels in the same electrophoresis tank (Invitrogen X cell II), one for transfer to PVDF and one for Coomassie staining. Not only does this show me how well my proteins have separated, it also acts as an alternative loading control, as per: http://pubs.acs.org/doi/abs/10.1021/pr1011476
I think that your protein quality is decreased, change the sample buffer ( there was problem in sample preparation process) anyway, you read "Western Blotting protocol" which included troubleshooting, that is comprehensive:
http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-us/service-and-support/handbooks/
I think there is a big difference between the running and transfer buffers. You should equilibrate the gel in the transfer buffer before transfer. Enlarging gel during transfer suggests something similar. Mightbe meoh is a bit too much, too. Try to lower to 10%. What is the pre-cast gel type?
yes they are out of date from Oct 2013 but previously I had used out of dated ones and nothing was wrong with that
Check the pH of your buffers.
The protein quality can be seen by how it runs, it is a smear or clear, discrete bands? A smear indicates protein degradation. Samples should be aliquotted to avoid repeated freeze thaw cycles and protease inhibitor cocktails used.
I have checked the ph of transfer buffer which is around 8.9 and running buffer which is 7.6.. Can you please tell me do I need to adjust the PH of buffer as I am using a company manufactured one I just dilute it to 1X concentration and the product detail doesn't say anything about maintaining PH
It looks like your proteins are degraded - the Bradford method will still detect degraded proteins, but when run out on a gel you get a smear like you are seeing here. Also your markers appear very faint, so you ma be having issues with transfer. I use 20% methanol in my transfers and have no issues, so I do not think it is the methanol. You may want to try a different transfer buffer.
tris-glycine system, running and transfering buffers both are pH 8.3 if i remember well.
@Azaz khan: please change the Tris buffer 1.5 (PH=8.8) and 1 M (PH=6.8) in Resolving and stacking gel, respectively.
It could be possible that your proteins are degraded, especially if they've had many freeze/thaw cycles. Although storage at -80C shouldn't have any impact on degradation. Biorad has a pretty thorough protocol for WB, including transfer and detection of bands:
http://www.bio-rad.com/LifeScience/pdf/Bulletin_2895.pdf
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