Hello,
I've been lurking on this site for all my protocol needs, and before I waste any time I figured I would run this by you guys.
First off I am an undergrad. who has exclusively worked with bacteria. However, now I am in a much different lab that focuses on plants.
I need to do a 'simple' total protein purification, so I started doing loads of research and here is what I found...
Many procedures use TCA, but I'm not sure if I have that chemical at my disposal. I do have acetone and ethanol and I have found many protocols that use either of these chemicals to precipitate proteins. My question is this...I guess.
I know that I should use protease inhibitors to protect my proteins, but when would I add them?? All of the protocols call for 4X acetone or 9X ethanol added to my crude extract after homogenization, but nowhere do I see that I use any special buffer with protease inhibitors. Could I add them straight to the crude extract?
I appreciate any advice.
-BioNoob22
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3 replies to this topic
#2
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Enthusiast
Posted 15 June 2011 - 05:30 AM
I would add the protease inhibitors immediately after protein extraction to the crude protein. Be forewarned, sometimes it can be extremely difficult to re-suspend samples that have been crashed out with acetone.
Lab Technician at Protea Biosciences
#3
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an elder
Posted 15 June 2011 - 10:32 AM
i would add the protease inhibitors to the buffer that will be used for the extraction immediately before use (some inhibitors are labile).
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#4
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Posted 17 June 2011 - 09:30 AM
What are these samples for, SDS-PAGE analysis?
If you're precipitating all the protein with TCA/EtOH/Acetone, you shouldn't really need inhibitors, you're going to be pretty much killing everything with those.
If you are just preparing these protein samples to run on gels, you can just do the extraction in sample buffer containing SDS and go straight to a gel.
If you're precipitating all the protein with TCA/EtOH/Acetone, you shouldn't really need inhibitors, you're going to be pretty much killing everything with those.
If you are just preparing these protein samples to run on gels, you can just do the extraction in sample buffer containing SDS and go straight to a gel.
