Hi everyone,
Been doing western blots for years now, recently however, whenever I run 2 gels, one of the gels would have a few lanes that exhibit wavy or smudgy bands at around 50 kDa (see images). Everything upwards is fine. The other gel however, displays great bands at the same MW region. I have tried using buffers from fellow colleagues and their equipment such as gel tanks and power packs and I still have this problem. Staining with Ponceau S can reveal the existence of the problem to suggest it's either a running or transfer issue and I have so far exhausted as far as I know, most troubleshooting options such as running buffer pH and using fresh buffers. Both gels run on SDS-PAGE properly without any visible problems at 90V throughout, with a semi-dry transfer (Turbo from Bio-rad).
My running buffer (1X) is composed of 25 mM Tris, 192 mM Glycine and 0.1% (w/v) SDS. For lysis buffers I use RIPA.
Any input or solutions from this great community would be great!
It looks like a transfer problem with the protein coming out of the gel but then migrating towards the electrode between the gel and the membrane rather than through...and then sticking to the membrane
you have two semi-transfer cases, always get one good and one bad. did you label the transfer cases as A or B, does bad band is always from one case?
Air bubbles between the gel and the filter, or insufficiently tight pressure between the gel and filter, are the usual reasons. Sometimes it's a defect in manufacture of the gel.
There must be two possible reasons for the problem your facing either gel is not formed properly or while transferring protein from gel to membrane some air bubbles might be present in between your gel and membrane.
For this, you should have run another gel as control and put it for CBB stain to ensure your proteins must separate properly. You should also take while placing the gel for blot transfer, make sure there would be no air bubble in between your gel and membrane, press it properly to remove air bubble if present any. Also, do the transferring step in an ice-cold transfer buffer and maintain low temperature throughout the process.
Before going for antibody treatment, stain your membrane with ponceau dye to check the proper transfer to the membrane.
All the Best.
You can stain your gel reversibly with Zn/imidazole before blotting to check if it ran ok. High salt in a sample, for example, can cause aberant bands. Another possible reason is that the upper tank buffer is too low (slow leakage).
Once you have confirmed that the gel is ok, unstain it with EDTA and assemble the transfer cassette. I do that on a small plastic tea tray, so that I can use plenty of buffer and thereby avoid traping any air bubbles.
Paul Rutland Junjie Shao Hi Paul and Junjie, thanks for your inputs, I will try using a different transfer case since I've been sticking to the same one (A). It's just weird because both gels are transferred on the same case and one of them exhibits this problem!
Hi Arnold,
Probably a silly question but just checking you are using deionised water for your buffers?
It is strange that it happens in just one area of the blot. If you have already tried fresh buffers I would think maybe a transfer issue. You mention this problem when you run two gels, is it always the same order of gel that has the issue? For example, if it is always the second transferred gel that displays this problem.
You say both run on SDS without problem but have you tried Coomassie staining to see if the issue can be seen? Then you would know if it is running or transferring.
Sorry can't be of more help.
Niamh
It should be something related to the transferring process. You should be careful in placing gel, membrane, and sponges (pads). Air bubbles or excess buffer remaining between the membrane and the gel would cause some problems.
Besides, use the appropriate sponges and, if needed, change them.
The gel is also fragile; if you press it more than enough, this might squeeze the gel, getting strange patterns of bands.
Check the followings:
1) Is the down edge of the gel bubble-free?
2) Have you properly removed all bubbles between the gel, membrane and up/down sheets?
3) Are the stacking gel and resolving gel formed firmly and in a straight line?
4) Are you loading the samples properly?
5) is your apparatus working properly, without overheat?
If the answers of all the above questions are yes, then you should not have the same problem anymore.
Hi Arnold,
I had the exact same problem with the Turbo blotter from Bio-Rad and I fixed it by changing the transfer protocol (~ 1.0 A @ 25 V const. for 30-40 min instead of the 7 min Turbo setting with 2.5 A). It seems that with the Turbo settings the transfer buffer can be heated up too much so that air bubbles are formed during the transfer process, which will lead to the exact pattern that you see on your membranes 2 + 4.
This issue is even described in the Bio-Rad Western Blot troubleshooting section ("Swirls or missing bands; bands appear diffuse on blot -> see also Blot Background > White spot"): https://www.bio-rad.com/de-de/applications-technologies/western-blot-doctor-protein-band-appearance-problems?ID=MIW4Q8C4S#blurrybands
Hope that helps - CL
We use a roller on the filter paper-membrane-filter paper "sandwich" to get rid of bubbles. It looks like you had an air bubble. Make your own gels to make sure that they are good.
- Badges
- Science method