- I make 10% SDS page gels and run at 110 v for 1.5 hours
- My marker separates initially however as it progresses the top MW markers disappear (the ones I need!)
- I have tried different % gels, new reagents/remade solutions, new SDS page set up, different power packs, different voltage, different buffers, different markers.
- When I run two gels, one gel appears to migrate fine and another one can be completely skewed, even when they are in the same casket.
- Under ponceau staining, you can see protein in all lanes even where the prestained marker disappears.
Usually higher MW markers are faint compared to low MW. You might use different brand of prestained markers or load larger volume. If you cannot see it after transferring to nitrocellulose, it could be that the transfer was not sufficient and usually low MW proteins are transferred better. If you have problem with transfer of two gels in same apparatus (the transfer is not similar) please make sure that the temperature is low (do it in cold room), cold buffer or ice bucket. Also check you transfer buffer.
It is quiet possibly the protein ladder you have been using have undergone many freeze-thaw cycles or have not been stored properly. The buffers in which they are reconstituted in may vary in their tolerance to freeze-thaw cycles. Same thing apply if they are not stored properly. I could only think of this as you see no issue with protein in all other lanes.
Hello Anya
I feel that the protein ladder you are using is degraded. The protein ladder has a limited shelf-life unless and until certain additives are added to lengthen the shelf-life like sodium azide (anti-microbial agent), dithiothreitol (DTT) (reducing agent), etc. Also repeat freeze-thaw cycles as mentioned by Vinod decreases protein stability. Protein stored frozen must be prepared in single-use aliquots in order to avoid freeze-thaw cycles.
Thank you.
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