5 replies to this topic

[biocrazy]

dears,

I expressed a protein with GST fusion. When I extracted the protein and checked using SDS-PAGE, I saw the band corresponding to the expected size of 48 kDa (22kDa + 26 kDa), but when i purify the protein using GSTrap (GE healthcare) I could not find the protein is eluted in the elution step (I checked the elution fractions using the Bradford reagent from Biorad).

Could anyone have troubleshooting for this situation.

Thanks a lot in advance, for your reply!

[mdfenko]

did you look for your protein in the load and wash fractions? maybe it didn't bind.

is the column fresh or used?

if used, has it been properly cleaned and regenerated?

[biocrazy]

mdfenko, on 01 December 2010 - 08:05 AM, said:

did you look for your protein in the load and wash fractions? maybe it didn't bind.

is the column fresh or used?

if used, has it been properly cleaned and regenerated?

Thank you very much for your reply!

I used a fresh column. The load fraction (unbound fraction collected while running the protein) had protein content. But it may be the mixture of many proteins. How can I know whether my protein of interst is there in the fraction. You mean, should I check by SDS-PAGE?

The wash fractions did not show intense color formation after adding Bradford reagent(another thing is I collected the washing fractions in 50 mL tubes, so it may be diluted i guess).

I will be waiting for your advice.

[pDNA]

Do you have an antibody for detection of your protein of interest?

Did you checked all your buffers for correct pH?

GST can  be a real pain ...is there a cleavage site to remove the tag from the protein? ...maybe the GST is already cleaved intercellulary and there is no chance for purification since the tag is already gone?

Is the size of your protein really correct? ...does it correspond to the site of GST+protein of interest?

Regards,
p

biocrazy, on 03 December 2010 - 03:58 AM, said:

mdfenko, on 01 December 2010 - 08:05 AM, said:

did you look for your protein in the load and wash fractions? maybe it didn't bind.

is the column fresh or used?

if used, has it been properly cleaned and regenerated?

Thank you very much for your reply!

I used a fresh column. The load fraction (unbound fraction collected while running the protein) had protein content. But it may be the mixture of many proteins. How can I know whether my protein of interst is there in the fraction. You mean, should I check by SDS-PAGE?

The wash fractions did not show intense color formation after adding Bradford reagent(another thing is I collected the washing fractions in 50 mL tubes, so it may be diluted i guess).

I will be waiting for your advice.

[mdfenko]

biocrazy, on 03 December 2010 - 03:58 AM, said:

I used a fresh column. The load fraction (unbound fraction collected while running the protein) had protein content. But it may be the mixture of many proteins. How can I know whether my protein of interest is there in the fraction. You mean, should I check by SDS-PAGE?

yes, run it on a gel to see if your protein is present in the mix and if it is at least partially depleted (if not then it didn't bind).

Quote

The wash fractions did not show intense color formation after adding Bradford reagent(another thing is I collected the washing fractions in 50 mL tubes, so it may be diluted i guess).

I will be waiting for your advice.

the elution fraction may also be diluted beyond the range of the bradford. you may want to try concentrating the fraction (spin filter concentrate on amicon or microcon) and also running it on a gel.

[biocrazy]

mdfenko, on 03 December 2010 - 09:08 AM, said:

biocrazy, on 03 December 2010 - 03:58 AM, said:

I used a fresh column. The load fraction (unbound fraction collected while running the protein) had protein content. But it may be the mixture of many proteins. How can I know whether my protein of interest is there in the fraction. You mean, should I check by SDS-PAGE?

yes, run it on a gel to see if your protein is present in the mix and if it is at least partially depleted (if not then it didn't bind).

Quote

The wash fractions did not show intense color formation after adding Bradford reagent(another thing is I collected the washing fractions in 50 mL tubes, so it may be diluted i guess).

I will be waiting for your advice.

the elution fraction may also be diluted beyond the range of the bradford. you may want to try concentrating the fraction (spin filter concentrate on amicon or microcon) and also running it on a gel.

Dear pDNA and mdfenko,

          Thanks for your advice. I will try your suggestions.