My lab is going to develop the protein technology recently
Our strategy is quick simple
extract to total protein from the sample and than do trypsin digestion and do LC-MS/MS to determine the peptide (our sample is from the lab-scale bioreactor)
and there is our extraction approach
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The biofilm samples (sponage) were frozen in liquid nitrogen and grounded .
The powder was mixed with 1 ml of a lysis buffer , 25 l of dithiothreitol (DTT; 1 M, pH 5.2), and 10 l of a protease inhibitor and was
subsequently subjected to five cycles of freeze-thaw treatment. The total protein was stored in acetone at 20°C overnight.
The protein was precipitated through centrifugation, and resuspended in a rehydration buffer(fresh) containing 7 M urea, 2 M thiourea, and 4% (wt/vol) CHAPS by centrifugation in 4°C 10000 g
( https://aem.asm.org/content/79/1/105.short )
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The problem is sometime I can't let the precipitated protein resuspended and dissolved completely
Is there any skill or different buffer that can improve this situation ?
I have try the cycle of pipetting and centrifugation but it seems not work
Chao-jui
sincerely
How do you know that the undissolved material is protein, rather than other components of the biofilm?
Try replacing the urea with 8M guanidine-HCl or guanidinium isothiocyanate, more powerful chaotropes. Also CHAPS is a relatively mild detergent. Try using SDS.
I think you will have trouble with the downstream processing (proteolytic digestion and LC-MS) if you don't remove the detergents and denaturants first.
Hi,
Why not try to sonicate your pellets, for example 5sec, 1 pulse(45% amplitude), it will help you dissolve your pellets in your resuspended solution.Thereafter, centrifuge at 20000g for 10-15min at 4degree.It is better to sonicate right before the first wash.
All the best!
Thank you for your suggestions
Actually I don't know the unsloved material is protein or not.
I centrifuge my sample in 10000g 4 degree twice to sperate the liquid snd solid after the freeze-thaw treatment,and than I add acetone to the supernatant in to the new tube, so I think the suspended solid were remove.
And before I added the acetone the liquid is transparent, after I added, these pellet suddenly appeared, so I think most of them are protein
Is there any possible candidate that could be precipitated by acetone ?
And is there any suggestion to evaluate the residual pellets are protein or other materials?
All the best!
The trick is to get quantitative information, so that you can determine what portion of the pellet is protein. You should wash it with water, dry some of it thoroughly, and weigh it. Then you can figure out how much of it is protein when you perform an analysis.
You might be able to get a protein measurement using the Lowry assay. The basic conditions can dissolve small protein pellets.
Amino acid analysis (AAA) would probably work on the pellet if it is protein because it involves heating the sample in HCl. If that doesn't dissolve it, nothing will. If it is protein, AAA will show lots of amino acids.
You can try dissolving a small amount of the pellet in SDS-PAGE sample buffer and running it on a gel.
Hi Chao,
In my sense, centrifuging is not a solution to dissolve. Did you try vortex? During my work I have used gentle vortex to dissolve proteins in my solution.
Good luck!
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