Beads pull down only free GST - (Mar/31/2015 )

Hey you all,

 

i am currently trying to express a protein in a GST-vector (pGEX6p1). I already cloned it successfully into the vector and the right frame etc is proven by sequencing as well as control digest. There are no stop codons within the protein or anything.

I did then the protein expression in Bl21 DE3 cells by using two methods: IPTG induction and auto-induction. For both methods i found out, that the protein is made via a dot-blot using anti-GST as well as anti-fusion protein antibody. Purification was done by batch purification with sepharose 4B beads.

Now here is my problem: when i load the elute from the beads on a SDS gel, there is only GST (26kDA band). No protein-GST band (which would be 70kDa). The protein i only find in the supernatant-without GST at its original size (42kDa).

I use lysis buffer with protease stop, so i don't get how this could happen?! Has someone of you had a similar problem? What i already tried was to do 2 purifications in a row, meaning that i incubated the supernatant/unbound fraction from the first round in a second. But even here i only have the band for GST and not the GST-protein.....

 

Any suggestions?!

Thank you all in advance!

 

 

Franzi

try incubating the beads with sds sample buffer to determine if the protein remains bound after elution.

 

if not then check the flow through and washes for your protein, your protein may not be binding or may be binding too weakly.

 

you say you use sepharose 4b but not what ligand is attached. sepharose alone is for size separations, not affinity. please give a more complete description of your isolation method.

Thank you for your answer.

 

It is Glutathione Sepharose 4B. I take the cell lysate, incubate it with the beads and elute the proteins from the beads. My problem is not, that my protein remains on the beads, the problem is that from the original protein-GST only the GST is eluted. The protein itself remains in the supernatant/wash etc. The problem seems to be, that the protein is cleaved off from the GST either before or during the batch purification, although there is no protease added.

are you sure the gst is cleaved? it could be masked.

 

there may be endogenous proteases present. do you add inhibitors to the lysis solution?

Yes my lysis buffer contains protease inhibitors.

 

I think it is cleaved because when i load all the samples on a sds gel, i can only see a very big band for GST and not for the GST-protein combination and in the supernatant there is a band for the protein without GST...

do you use a inhibitor cocktail or do you add individual inhibitors?

 

some inhibitors, like pmsf, have a very short half-life in aqueous solution. do you add your inhibitors fresh immediately before use?

For each cell lysis procedure i make new buffer. Add the pmsf (from frozen stock solution) as well as the inhibitor cocktail immediately before usage. It is not individual inhibitors but a cocktail from Roche.

the frozen pmsf stock, is it in aqueous solution or alcohol (100% methanol, ethanol or propanol)?

 

if it's aqueous then it's probably decomposed. try fresh pmsf.

It's in isopropanol

Hi,

 

your Gluthadione Sepharose 4B is fresh or are you using a regenerated resin?