Protease inhibitors - (Mar/05/2007 )
I am looking for any receipe how to make protease inhibitors coctail.
I am serching for secreted bacterial proteins.
So far I have been working with coctail from Roche but at the moment I work with large volume (750 - 1000ml) and adding about 20 tablets is required what make it horrible expensive.
I thought about PMSF but is rapidly degradated in the medium (1/2 hour). I have to dialyse my medium for 4 days so it's not going to work.
I will be grateful for any suggestion.
Thanks very much!
Aja
I understood PMSF was good for a day, which could mean make it fresh for each buffer change.
I always used PMSF and EDTA for my stuff.
From my recall, if you can knock out the proteases early on, they tend to not be a problem, even if your protocol goes on for a few days. What does your protocol entail? Can you alter it to make the process more streamlined (ie quicker)?Why do you have to dialyse for so long? Don't forget that dialysis at 4C should slow down reaction too.
Hi Swanny!
Thanks for your replay 
I'm just optimising protocol and searching for information.
I haven't used PMSF yet, I just read somewhere but if it inhibits proteinase in non-competitive way it’s worth to try. Could you please tell me which concentration PMSF and EDTA did you use?
I will also try to dialysis at room temperature (I used 10C before). I try to remove casein enzymatic hydrolisate (0.5%) from the medium and it gives high protein background in protein assay so I can’t determinate the concentration of secreted proteins. The Amicon ultra filter (5kDa cut off) doesn’t work in this case.
I don't even mention about mess the casein hydrolysate makes on 2D gel.
I will be grateful for any suggestion.
Aja
PMSF and EDTA can be used at 0.1 to 1 mM
About the casein hydrolysate: I haven't used it. What kind of size fragments does it contain?
Other questions: How big is your protein of interest? What cells are you using? What is your current protocol?
Hi swanny!
casein hydrolysate should contain fragments no biger than 1kDa (0.4 - 0.9). I use casein and casein hydrolisate to growth Streptococcus uberis, which cose e.g. mastitis. I am doing a screaning of proteins which are secreted to the medium and (but it will be done later) attached to the cell wall. I think I am interested in proteins more than 10 kDa because I can detected them by electrophoresis.
Protocol so far:
centrifuge ,
filter ,
dialysis (3-4 days at 6C - can be shorter),
centrifuge with Amico Ultra 5kDa cut off (to dense from 1000ml to 4-5 ml),
precipitation ,
resolve in 2% SDS - sds page, or in lysis buffer -2D
Thanks again for your help.
aja
casein hydrolysate should contain fragments no biger than 1kDa (0.4 - 0.9). I use casein and casein hydrolisate to growth Streptococcus uberis, which cose e.g. mastitis. I am doing a screaning of proteins which are secreted to the medium and (but it will be done later) attached to the cell wall. I think I am interested in proteins more than 10 kDa because I can detected them by electrophoresis.
Protocol so far:
centrifuge ,
filter ,
dialysis (3-4 days at 6C - can be shorter),
centrifuge with Amico Ultra 5kDa cut off (to dense from 1000ml to 4-5 ml),
precipitation ,
resolve in 2% SDS - sds page, or in lysis buffer -2D
Thanks again for your help.
aja
Seeing as how you are trying to purify from the medium, can you do an ammonium sulphate precipitation? You can look up the technique by Google, if you're not familiar with it. It's very straightforward, and can give you relatively clean protein in small volumes, in a fairly short space of time. Basically, you fractionate your total protein mix by selective precipitation that can be readily reversed; this should also help you get rid of the casein mixture.
Thank you Swanny!
I will do that. At some point I forget about ammonium sulphate precipitation.
I will let you know how it's going. aja
pmsf can decompose in as little as a half hour depending on pH (i think high pH destabilizes it, but i may be mistaken and it is low pH) but it still won't last a day in aqueous solution. it does, however, work quickly so that it won't be needed unless something else is added to the solution.
pmsf can decompose in as little as a half hour depending on pH (i think high pH destabilizes it, but i may be mistaken and it is low pH) but it still won't last a day in aqueous solution. it does, however, work quickly so that it won't be needed unless something else is added to the solution.
I work with pH=7.1 so 1/2 hour should be enought to knock out potential proteases.
PMSF's just come so I will be test it next week.