Hi all,
I am having a very odd issue regarding my Western blot transfers. When I perform a transfer, sometimes my bands diffuse across huge areas of the membrane and become 'wave-like.' I've attached an image of a Western blot showing this issue.
I have spent hours sifting through Western blot troubleshooting guides but I was unable to find this problem described anywhere. I believe I can rule out the following issues:
1. Diffuse bands resulting from too much protein. I have overloaded Westerns before and have seen what it does to the bands; the current problem is distinct because the bands are no longer bands, rather they appear like waves across the membrane.
2. Insufficient contact between gel and membrane inhibits transfer. Initially, I thought this was the source of the issue however, bubbles produce circular voids of signal. With my issue, the protein is indeed being transferred but it is not being retained in a band.
Another observation that adds to the odd nature of this issue is the frequency at which it occurs. I only see this effect for ~25% of the Westerns I perform. In fact, just yesterday I ran two transfers in the same chamber - one transferred perfectly while the other had this issue. I believe this rules out the buffer and/or the transfer equipment as the source of the problem.
Any and all feedback is greatly appreciated! This problem is becoming increasingly frustrating...
Thank you for your time,
Ryan Chaparian
Did this happen with the same transfer cassette? If yes, try to check if the cassette is becoming too loose / if the sponge deformed / if you put enough filter paper layers into the sandwich. Make sure the cassette is tight enough but not too tight when you are putting it into the transfer tank.
Dear Ryan Chaparian ,
I think you have to recalibrate the power supply unit. Also, prepare transfer buffer freshly every time.
Next time, run two PAGE simultaneously and one should proceed for western and other one for CBB staining.
hope it will solve you issue.
Try to ensure that there are no any air bubble in the contact point and also in the sample loading points. Also try to decrease the voltage during the transfers.
are you sure that this is a transfer problem Ryan?
It looks like samples running outside of the gel as well as through the gel in the initial gel electrophoresis run to me. Do you notice any loading dye moving oddly in the gel running stage? Is it always the same position samples in the gel?
@Anil Gupta: Sometimes when I run two transfers simultaneously in the same chamber, one will transfer well and the other will not - because of this, I believe it is not an issue with the power supply. Also, I use fresh buffer every time so I don't think that is the issue either. However I will try Coomassie staining to see if the protein is running weird through the gel; thanks for that tip!
@Binayak Raj Pandey: I'm fairly certain there are not air bubbles between the gel and membrane (I use a roller very thoroughly when preparing the sandwich). Regarding the volts, we use 10V constant for 60 minutes as this seems to work well for others in my lab. Would you recommend going lower than that?
@Paul Rutland: Interesting assessment, my pre-stained protein ladder and loading dye in my samples always migrate normally (in fact, for the Western I've attached in my post, the dye front was very crisp). As I've been having the issue for months, I've run many gels and have rotated the sample positions on the gel so I do not believe that is the source of the issue.
Based on some of the responses, I decided to investigate the migration of protein bands through the gel. The logic behind this being, if the protein runs aberrantly through the gel, it could produce the banding pattern (the wavy-ness) I'm observing with my Westerns.
I loaded a single 15% SDS-PAGE gel with multiple lanes of my sample and ran the gel at 160V for 60 minutes. Then I cut the gel in half and proceeded to transfer one half onto a nitrocellulose membrane and while silver staining the other half. The transfer performed poorly again the wave-like distribution of the protein onto the membrane was apparent from the ladder sample. However, the silver stained gel (image attached) shows that the proteins migrate through the gel normally. Thus, I can rule out that the source issue occurs before the transfer is started.
Thank you all for your feedback, if you have any other ideas I'd love to hear them!
One hypothesis would be that your membrane got old. I´ve had that issue once. While equilibrating the membrane, check closely if the whole membrane becomes evenly hydrated. If you see spots with a less translucent appearance, there are big chances that your transfer will fail.
Did this happen with the same transfer cassette? If yes, try to check if the cassette is becoming too loose / if the sponge deformed / if you put enough filter paper layers into the sandwich. Make sure the cassette is tight enough but not too tight when you are putting it into the transfer tank.
@Man Hei Cheung: This was the source of the issue!! After reading your response I inspected our sponges and compared them to an unused sponge - the ones I had been using were very compressed by comparison. After using extra filter paper to compensate for this, my Westerns have begun to transfer well again! THANK YOU!
@Harumi Ito and Patrick Jack Spencer: Thank you for your responses, I have also started soaking my membrane in transfer buffer for a longer duration to ensure efficient equilibration. I think it definitely facilitates a better transfer. Thank you for this tip!
Thanks everyone for your input, this has been extremely helpful!
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