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GST Extraction: Protein Denatures???


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#1 mikelb7

mikelb7

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Posted 22 October 2010 - 09:42 AM

Hi everyone,

I've been trying (unsuccessfully) to purify LIF (leukaemia inhibitory factor) from a pGEX-2T vector (in E. coli), using glutathione agarose beads. LIF is supposed to be soluble AND stable at 4 degrees (as it is added to the media of ES cells so they do not differentiate).

Anyways, the pGEX vector places a thrombin cleavage domain, and the GST (glutathione-s-transferase, the tag) genes downstream of the protein of interest (LIF), and protein production/construct stimulation occurs with IPTG. The resulting fusion protein is therefore a thrombin cleavage domain linking LIF to GST. The protocol I'm using (very similar to http://www.mshri.on....cility/LIF.html) calls for the binding of GST-LIF to glutathione-coated beads (as GST binds glutathione). After this, thrombin is added to cleave LIF from the GST-Glutathione bead complex and centrifugation/washings further elute LIF from the bead complex.

After running through this protocol 4 times, I am unable to generate significant amounts of contamination-free LIF. After taking diagnostic samples (and subsequently running them on SDS-PAGE), I have determined that the thrombin cleavage step (end of Day 5 on the linked protocol... pretty much the 2nd last step) must be the culprit. Before adding thrombin overnight, SDS-PAGE shows a relatively large amount of GST-LIF fusion. Following the thrombin cut, the GST-LIF fusion band is no longer visible. This is accompanied by a great increase in the amount/concentration of free GST, while unfortunately (for me), LIF is present in an almost undetectably low level. The resulting washes/supernatants contain very little fusion or LIF, yet lots of GST tag still.

Has anyone ran into problems such as this before with Gex-extractions??? A colleague of mine has recently ran through this experiment himself with relatively no difficulty, yet aside from filter sterilizing all my solutions (which he did not even do...), we are out of further optimization/improvement ideas. I therefore must defer to BioForum's collective expertise :)

Thanks in advance for any/all help and I'd be more than happy to clarify any of the protocol steps/the methods I'm using if I haven't been clear enough.

Mike




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