Dear Annie Aglin

some questions to understand your problem:

1) is it your protein a membrane protein containing hydrophobic regions?

1) Did your protein containscisteines? if Yes, how many and Which reducing agents did add in the loading buffer? (BCE, DTT, TCEP?)

Best

Manuele

1) It is not a membrane protein. The protein has 57% hydrophilic amino acids and the remaining are hydrophobic.

2) My protein doesn't have any cysteine amino acid. I used DTT as a reducing agent in the loading dye. I have also tried purifying the protein using an 8M urea buffer.

Sequencing the band is the only way to know if it is your protein. You can do an anti-his western blot to see if the His-tag is exposed or buried. After confirming that it's your protein, you can consider why the size is off.

Also depending on the strain of E. coli you used, there are some endogenous proteins that are overexpressed upon induction. Do you have a positive control (a gene of a protein that is expressed well of a different size, cloned in the same plasmid using the same E. coli strain)? If so, you should compare the background of the gel to see if this is one of those proteins. The background you should compare is whole cell uninduced and whole cell induced.

Hello Annie, how wad this protein cloned? It is possible that you have transformed E. coli with the incorrect plasmid. Your protein may also migrate aberrantly or form oligomers that do not dissociate upon boiling with SDS-PAGE. As a first step I would re-transform with the plasmid and if the issue persists I would sequence the plasmid. Good luck!