DEar Fatin Safiudin

can you share with us the sds-page picture and some more indormation about your protein? Comparision of induced vs non induced sample showed a clear induction band at the correct mw?

best regards

Manuele

Unless you have an extremely high % of his tagged protein in the crude extract you cannot reliably assess binding to the column based on A280 of the extract and flow-through. I would echo the comments of Manuele Martinelli, as answers to these questions will inform you whether you have an issue with protein expression or with the chromatography step.

Do you have evidence that you are in fact expressing your protein of interest? More information about your expression system would also be helpful to aid in troubleshooting.

Your description suggests that you compared the Load sample with the Flow-through sample by A280 spectroscopy. In the first place, even if you do get sufficient expression, the difference in protein amount between the two samples would perhaps be negligible and undetectable. Check your expression level, you might have not expressed the protein of interest. Since multiple proteins amount for the A280 reading for both samples, A280 spectroscopy is not appropriate, it is appropriate if you measure a single protein and know its extinction coefficient. A more appropriate method is a colorimetric method such as Bradford or BCA.

You must take samples throughout the whole process to know that.

Gather the flow through, the washing fractions, etc, and run an anti-HIS western blot.

Then you should know at which step you lose your membrane protein.