Hi,
I am looking to precipitate and/or concentrate a protein (around 45 kDa) from a gram-positive bacterial culture supernatant. The media used is GM17, which is fairly complex with loads of protein hydrolisates, which I don't know if will interfere with the extraction. The protein will be used for Western blot quantification, so it doesn't need to retain activity and can be denatured.
The estimated amount of protein in the supernatant is fairly low, around 1 ug/mL, so I guess we'll need to use large volumes of supernatant to isolate an amount compatible with western blotting.
We've tried with ultrafiltration, with Microcon columns, and cold ethanol precipitation, with no luck. I'm aware of several precipitation methods, such as phenol, chloroform/methanol, TCA or ammonium sulphate. I don't really know if the protein is degraded during the culture, which is carried o/n at 30ºC, and if this is the reason why we don't see any protein.
What can we do to extract this protein?
Thanks!
Is there any scope to generate a version of the protein with an affinity tag on it? This would then allow you to batch bind or pass the supernatant through a resin bed, pulling the protein out of solution and concentrating it up from a large volume, eluting into a smaller volume for the Western. Secondly, if you can only use the untagged sequence, what is known about the protein? - if you have the sequence it may be possible to look at things like the charge (pI), hydrophobicity, structure in case it may be captured by some of its parameters etc.
Good luck.
Can you decrease the volume of the supernatant and therefore increase the concentration of your protein of interest in the supernatant?
Alternatively, how good is your antibody? Can it detect very small amounts of your protein? I run frequently blots with less than 1 ug protein and use either Pierce Supersignal West Dura or Biorad Clarity for detection and get strong signals.
Another question is whether your protein could have been digested during your protocol of enriching it. Can you include a protease inhibitor cocktail?
Good luck!
During precipitation several proteins escape and therefore you may not be able to get all proteins, but try acetone+TCA. Keep acetone at -20 and use 10-20% TCA. precipitate for 30min-1hr on ice and centrifuge at 15000g for 1 hr.
Thanks for your answers. We've tagged the protein with GFP, to allow its detection by immunobloting (the antibody, from Clontech, can detect as low as 5 ng, which is quite good). I haven't been able to find a suitable antibody for the target protein. The aim is to precipitate the fusion protein from the bacterial culture medium, so it's not really important to purify it.
I still haven't tried the acetone/TCA and the chloroform/methanol protocols, our lab is not really focused on proteomics and we have to buy most reagents before trying a new protocol. For now I'll try both methods and depending on the results will eventually move to affinity chromatography.
Thanks!
If I'm not mistaken GFP sticks very effectively to Hydrophobic Interaction Chromatography resins if you get the conditions right. This could be a viable - and easily visible - means of bulk-concentrating up your fusion protein if it is present in the supernatant.
There is a paper on ResearchGate which describes exactly this :
Purification of recombinant green fluorescent protein from escherichia coli using hydrophobic interaction chromatography
Article in Journal of Liquid Chromatography & Related Technologies 37(13):1873-1884 · April 2014
Mohd et al - PDF link below
http://www.tandfonline.com/doi/pdf/10.1080/10826076.2013.825847
Good luck.
Hi Robin, I wasn't aware of this technique, will definitely have a look. Thank you!
hi Aleixandre , i use the same media (GM17) for expression my target protein, i used the cold actone for precipitation of my protein at culture supernatant ,its a good and simple method , when i test the presence of my protein in the pellet with DOT BLOT method i detect the protein , but when i load it on SDS PAGE GEL , the sample was moved horizontal , and push other sample. i think maybe the acetone has the effect on movement the sample on sds page.
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