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GST Purification Protocol and usage of Factor XA issue

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#1 manchestermicro



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Posted 23 February 2011 - 03:09 PM

Hiya everyone,

Hope all is well!!

I am currently trying to purify a GST tagged protein (first timer with GST) that is in PET41a. Growing it up and inducing it is seems to go very well.. However, when I get to the purification step there is a bit of an issue..

My last SDS Page and westerns showed
1) a band the size of my protein and gst together
2) cleaved protein
3) my protein

So I pooled together my elutions with the highest signal and put them down the column again to clean them up and my protein purified showed in the flow through column{ I have no idea where the other bands went as it was alone!!!! And there is nothing in the wash.factorxa or elution columns} Sadly though there is not enough of the purified protein to place in a AKTA/heparin column. So I need more.

With all that said I am doing it over again!! if anyone has any suggestions to this protocol as this was made up by a student before me (I am also going off Qiagen as this is the most similar to the one I was given) as follows as... I have very little experience with GST it would be much much appreciated!!

Column contains 2 ml resin(the volume of resin used is a column volume)
Cleaned with 5 column volumes of distilled water.

1) Equiliberate column with 5 column volumes of binding buffer (TRIS HCL 50mM/Nacl 50mM ph 6.8/6.9)

2) Cleared Lysate this is from about half a liter so after the french pressing and such I end up with about 11-15 mls (I am not sure on the amount to add to each column of resin)..

I am thinking about dividing this up.. Maybe 3 mls for each column as to not oversaturate.. Any thoughts would be much appreciated! Mix this up let it set for 30 minutes.. Collect the flowthrough

3) Wash the column with my binding buffer (TRIS HCL 50mM/Nacl 50mM ph 6.8/6.9) (20 column volumes)

4) Cap column and add Protease (FACTOR XA) Previously I had added 1 ul of protease into 500 ul of binding buffer.. I am not sure how to calculate this if I decrease this to only 1 ml As the protocols I have read regarding this say that it needs to have 10 ug/ul to a 40ul reaction??? or needs to be at least 0.25mg/ul not sure about this neither is my supervisor... Left at 37 degrees overnight. Then collected..

5) Then 10 elutions (500 ul of binding buffer TRIS HCL 50mM/Nacl 50mM ph 6.8/6.9)

Any help would be great I am not sure how else to modify this anymore..

Thanks for any replies!!


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