Hi everyone,
I just began running my western after loading my lysate samples, and 20mins into running the gel some of my samples look like this.... (see attached graphic).
Some additional details: I've purchased the gel precast, and the gel has not dried out/expired. I run at 80mAmps for 45 mins (however I begin seeing this 10 mins after running my samples). I also flush out the wells (from the storage buffer) before loading my samples so they lay evenly.
Is the issue with my laemmli buffer composition to lysate? Pipetting enough before loading the sample? Or should I boil my sample for longer before loading? Thank you in advance for any replies.
Since your protein marker is running fine, I don't think the fault is in the gel. I have not seen such a pattern of dye front previously. Can you share what buffer you are using in the tank? Is it normal Tris-Glycine-SDS buffer or are you using something else? Were your samples too viscous and what is the quantity of protein loaded into the wells?
Hi there Tamilselvan,
I agree, I don't think the problem is the gel. I use Tricine SDS running buffer, however, our lab re-uses running buffer, so it had been used one time before I ran this gel. Also, my samples could be vicious, I study lung epithelial cells that produce mucus (I also lyse my samples with 2x RIPA). However, I do spin down my samples and collect the aqueous supernatant for the lysate prep. I loaded 20ug of protein per well.
Thank you for helping me troubleshoot this!
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