I obtained two peaks during the affinity chrokatography which are next to each other. After I run SDS-PAGE, both peaks are my target protein. I am curious about the reason behind that, can anyone explain this?
Thank you!!
Hello, several reasons may cause this.
- Make sure you do not overload the column as this would compromise the resolution capacity of the run for proteins that are nearby.
- If you use a N-terminal His-Tag the contaminant could also be a truncated version of your protein and thus gets eluted in the affinity.
- May be your protein contain any disulphide bonds. If so it is possible that there is a proportion that has been proteolytically cleaved yet the protein remains held together by the disulphide bonds.
Make sure you are completely reducing your protein during the purification and with SDS-PAGE sample buffer. Unreduced disulfide bonds could affect the apparent molecular weight on SDS-PAGE.
- Did you concentrate your protein after the all purification steps and if yes,did you see this extra band before concentrating your protein or after that.
Good luck
Hello, several reasons may cause this.
- Make sure you do not overload the column as this would compromise the resolution capacity of the run for proteins that are nearby.
- If you use a N-terminal His-Tag the contaminant could also be a truncated version of your protein and thus gets eluted in the affinity.
- May be your protein contain any disulphide bonds. If so it is possible that there is a proportion that has been proteolytically cleaved yet the protein remains held together by the disulphide bonds.
Make sure you are completely reducing your protein during the purification and with SDS-PAGE sample buffer. Unreduced disulfide bonds could affect the apparent molecular weight on SDS-PAGE.
- Did you concentrate your protein after the all purification steps and if yes,did you see this extra band before concentrating your protein or after that.
Good luck
may be you have another native protein have multi histidine residue , so try to make separation between them and do SDS-PAGE for confirming what is your band.
Also for insuring you can proceed western blot, ELISA, ....etc
best regards
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