Hello everyone, I have condition in my sds page tricine here. My sample is recombinant human epidermal growth factor (protein 6,2kDa). before going to sds, all the sample have to be extracted (periplasmic extracted). pic attached here (under). can you tell me whats probably the problems in my sds? thank you for your responses
Is this a stained gel, or is it still running? It looks to me like it is a running gel, and the dye extending down the edges of the lanes is the loading dye. This may have happened because the sample volume was large and there was a high concentration of detergent or glycerol in the sample.
Mine is sds page tricine schägger.
Yes it still running. I will give you the final pict gel after destaining. But my sample volume is 20 mikroliter (15 microL for sample + 5 microL for buffer sample). How much concetration of glyserol that you used?
Indah; the gel looks overloaded with sample. Try loading half the amount.
Andrew: is it overload if i load 20microL sample in well?
Viji:
1. Yes, i load 20microL
2 and 3. Yes and i remove the comb gently
4. No as i remember
5,6,7: staining are mixture of coomasive blue, metanol, acetic acid and i use 30 V initial after the sample in through stacking is 100 V
Thank you for your responses. It means a lot for me :D
Indah, I think it depends upon the concentration of protein, more than the volume. 20 ul doesn't sound like too much in terms of volume.
Prepare new buffers and dilute your sample, I think you have a lot of protein and sds is not enough. You can add 20 uL to each well if you want.
Very poor resolution of bands is likely due to a problem with the buffers used to prepare the stacking and separating gels, as others have suggested. Pay particular attention to their pH, which is quite important.
All right. On my next sds page i will make a fresh buffer and pay attention to each pH. Thank you all :)
Yes there may be a problem with the buffer solution formulations . In addition , the blue band in the gel has a higher mobility at the left-hand end of the gel. Are the electrode tank buffer solutions in full, even contact across all of the gel.
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