Is the major band at the expected molecular weight of your protein?

Do you know that your protein is being expressed?

Interesting! Following answers

@adam

Sir , my protein is being expressed as I always get band n also after sonication I get my protein in soluble form too but when I go for purification I face this problem

If the protein is in an aggregated but soluble form, it may not bind well because the His tag is obscured.

If the protein undergoes proteolysis causing the His tag to be cleaved off, it will not bind.

Cut out the band from the gel and submit it to a protein sequencing facility to determine whether it is the right protein and whether it contains the His tag.

It is possible your percentage of glycerol is too high. try to load your sample twice and slowly wash Ni-column with binding buffer initially.

@xu zhang

I used 10%glycerol .. I will wash next time twice then.. and during binding I keep at rocker shaker at normal speed...not so high..not so low...

we usually pack the column, and use bio-rad pump load sample and wash the column. Glycerol can influence the binding of protein to the column.

@xu zhang

Thank you

I will try without glycerol next time .... If u get any idea please let me know in future.

If the major band corresponds to your protein of interest, then there is a lot of is in the flow through. So either the capacity of the column isn't high enough, or you need to improve binding conditions. Sometimes the His-tag is shielded inside the protein and can't interact with the column. In those cases, tagging the other end of the protein (C vs N-terminal) may help.