The protein size is 14kDa. I am purifying my protein by gel elution after excising the appropriate band. Previously, I have seen the single band at 14kDa position but after keeping it in 4 degree for a week, the protein band is now seen at 100kDa position. What the reason behind it? Does my protein getting aggregated and not moving properly from stacking gel or also got stuck in the resolving gel?
Your protein may have some Cys residues and after a week storage it can form oligomers by disulphide oligomerization. To test that you need to run a gel of your protein under non-reduced and reduced conditions. If this is a case, under non-reduced conditions you will see the band around 100 kDa and under reduced conditions the band about 14 kDa. It's also possible that your protein has a very hydrophobic nature and after a week storage it can form aggregates by hydrophobic interactions. To test this you need to add 6 M urea to your SDS-Sample buffer (with b-mercaptoethanol or DTT). That should cause dissociation of the aggregate and you will see it on gel.
Your protein may have some Cys residues and after a week storage it can form oligomers by disulphide oligomerization. To test that you need to run a gel of your protein under non-reduced and reduced conditions. If this is a case, under non-reduced conditions you will see the band around 100 kDa and under reduced conditions the band about 14 kDa. It's also possible that your protein has a very hydrophobic nature and after a week storage it can form aggregates by hydrophobic interactions. To test this you need to add 6 M urea to your SDS-Sample buffer (with b-mercaptoethanol or DTT). That should cause dissociation of the aggregate and you will see it on gel.
Hi, I totally agree with Viktor. You should check the Aa composition of yor protein to assess the idrophobic Aa. I suggest to you to split the sample in two an to denaturate your proten by using a chaotropic reagent (UREA 8 Molar or Guanididium Cloride 6 M) and run a gradient precast gel to better separate if some other things are present with your protein.
I have a curiosity: Do you still see some signal at 14 KDa?
#Viktor#Simone, Thanks for the valuable suggestion. I will try suggested approach and update the status.
#Simone, there is no signal at 14kDa. Band is only seen at 100kDa position.
A word of caution regarding Simone's advice: 6 M guanidine hydrochloride will mess up the electrophoretic migration of your sample (high ionic strength). Better stick to urea + SDS.
Yes, I agree better to stick with urea and SDS surely you will get band at 14 kDa positiion.
Just a question to Rupendra Shreshta- after addition of SDS & DTT (or beta-mercaptoethanol) containing loading dye, are you warming your 14 kDa protein sample over a water bath to aid the dissociation ? If you do it this way do you see the aggregation?
#Menon; Yes, I heat the sample in the heating block at 96 degree for 3 minutes. Previously it was on correct position after heating also but after storage it has a problem. Now the intensity of band is low as compared to previous as if degradation is happening. I am using PMSF and protease inhibitor cocktail in protein sample for storage but why degradation?
I think your protein did some cross-link with acrylamide or it is not yet properly eluted. Which method are you using?
Did you use reduced conditions in order to get your 14kDa protein from the gel? Is this a bacterial recombinant protein ? The question is just to make sure that it is not a glycoprotein! As you know the migration in PAGE is different!
How did you elute the 14 kDa from the polyacrylamide gel? Are you sure that elution do not modified the protein? Did you try to run the 100 kDa protein by boiling for 5-7 minutes the in a sample buffer with reduced conditions ( beta-mercaptoethanol and SDS)?
I'm sure all of the suggestions from the other researchers are very good and you should try all.
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