I have question regarding Native gel (15%). My protein molecular weight is 13 kda and it supposed to form dimer when ligand is added.The ligand is around 500Da. When I run native gel, my protein dint enter into resolving gel.I ran it at 70 V for 7 hours in cold room and observed that my dye front ran till end,
Attached is my native gel picture. With reference to my native gel, the first 3 lanes are my protein alone. It is strucked up and not enter the resolving gel. Moreover I understand if the protein sample struck on the well, It means its getting aggregated. so the part of my protein is aggregated but rest dint enter the resolving gel.
The lane 4 -6 are the samples with protein and ligand. It supposed to form dimer when it run. But I couldnot interpret anything from the gel.
May I know how can I interpret results from native gel ? How can I check the dimerisation using native gel ? Which recipe I have to follow ?
Difficult. Try SDS-PAGE at low SDS oncentration (0.1%) or native gel electrophoresis in 8-10% gels and see what happens. 15% is quite high.
Try NATIVE-PAGE (15%) and load protein ladder in one well so that you can compare pattern of NATIVE gel with your protein and before loading your sample take the absorption spectrum (320nm-220nm) of your protein in order to confirm whether Your sample is protein or other macromolecule and which method you are using for protein purification?
Can you use another method to test for dimerization? instead of native gels maybe size exclusion chromatography on a FPLC? or if you want to stick to gels - can you cross link your protein upon binding to the ligand and then load it onto the gel?
Hello Ya Yesh :) I think that, as Francisco Solano suggested, you should try with a gradient, I put a 4% concentrator and then a 5-12% gradient, since your protein is very small I would try either 8-12% or 8-15%. I run my gels at 15mAmp (2hrs) if it is a small one or 30 mAmp (4-5hrs) if it is big, you could read this paper, it could be helpful.
Blue native PAGE. Nature Protocols 1, - 418 - 428 (2006) Ilka Wittig1, Hans-Peter Braun2 & Hermann Schägger
Best regards!
Check the isoelectric point of your protein (Protparam). You could also determine how many negatively charged groups you expect to have (use a pKa predictor). It looks to me like you have a basic protein. If the pI is close to 8, you should try acidic native gels.
Dear Carol,
Yes. My protein PI is close to 10(calculated with Protparam, expasy). I tried in acidic native gels as well. But I can see more number of small bands (protein + ligand) compared to the protein alone. This occurs just after it enters the stacking gel. is it because my protein is denatured or possibly degradation ? Or it is because of ligand effect makes my protein likely to undergo degradation ?
Hi Ya,
I'm not sure, either protein degradation or a conformational change would explain a smaller band. Check your protein+ligand on SDS-PAGE. Does the ligand have a positive charge? I'm not sure what you mean by "just after it enters the stacking gel". When I run native acidic gels I run them for about 75 minutes at constant current of 30 mAmp. Hope that helps.
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