protein detected on SDS PAGE but not analytical HPLC

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usvganesh
usvganesh's picture
protein detected on SDS PAGE but not analytical HPLC

Dear Folks,

I am working with isolation and purification of protein of around 6kD produced in E.coli.

I am facing problem in its analysis i.e after IEX when I am running samples on SDS page I could see bands corresponding to size of my protein of interest. But when i am submitting samples for RP-HPLC analysis using C-18 column, I am not able detect protein of my interest. Where as standard / innovator samples appears to be fine in both the cases.

can anyone share experiences/ soultions

Ganesh

Sami Tuomivaara
Sami Tuomivaara's picture
Ganesh,

Ganesh,

You didn't tell about the method of detection of you HPLC or the gel staining, nor how much sample you load in your gel and the column.

If you detect by UV at 280 nm, your protein might not have (enough) tyr and trp residues to be detectable, it is totally possible with such a small protein. Also, refractive index detection is not very sensitive, here it depends how much protein you inject per run.

Other possibility is that your protein gets stuck in the column, usually C4 or C8 resins are used with proteins because the interaction of protein with C18 can be quite strong. You can try higher organic content (or gradient) in your elution buffer or C4 or C8 resin to solve this.

Cheers,

Add colour 2 ur life
Add colour 2 ur life's picture
Hi Ganesh,

Hi Ganesh,
I totally agree with suola. This might be the continuation to his answer ,volume you are loading onto SDS-PAGE is HPLC column is important factor,the detection method as well.

Balu..