I need to lyse insect cells (Tn5B lineage) to produce antigens using recombinant baculovirus. I need to use a protease inhibitor to protect my antigen. A researcher suggested that I use Halt ™ Protease Inhibitor Cocktail, EDTA-free (100X). However, it is very expensive and yields little reactions.
I found the Protease Inhibitor Cocktail I (Merck / Sigma) which is cheaper and yields more reactions. However, the protocol says to dissolve the powder in 10ml of RIPA Lysis Buffer, 10X (Catalog # 20-188). Thus, it would be necessary to buy another reagent.
Could someone who has worked with any of these reagents tell me if I really need to dilute the powder in the RIPA Lysis Buffer?
The lysis buffer I use contains:
20mM Tris HCl
300 mM NaCl
10mM Imidazole
1% Triton X-100
10% Glycerol
Could I not dilute the powder in my buffer?
If anyone has any suggestions, can you help me?
Hey Leonardo,
we use the cOmplete™ Protease Inhibitor Cocktail from Sigma.
Dissolve one tablet in 1 ml of H2Obidest and you get an 50X Stock. We aliquot this stock in 20 µl and store it at -20°C.
When we want to use it for lysates, we add the 20 µl aliquot to 1 ml of RIPA and and get a 1X concentration of the protease cocktail. And then your RIPA is ready to go.
Of course you can use any volume of RIPA as long as the dilution is 1:50 to get the protease stock to 1X (e.g. 1 µl in 50 µl RIPA or 100 µl in 2ml RIPA and so on).
All the best,
Marc
Hello Leonardo,
Our lab uses SIGMAFAST™ Protease Inhibitor Cocktail Tablets, EDTA-Free which can directly be dissolved into your buffer you listed.
I roughly use half of a tablet to 30ml of my Lysis buffer(quite similar to yours), depending on the cell mass you might need to use more, but that is generally been ok.
If you first crush the tablet in metal spoon to a fine powder, its much easier to divide and dissolve into your buffer samples.
Hope this helps.
With best regards,
Mikko
If the cocktails are too expensive for you, it's probably a good time to ask a simple question: What's the evidence that your protein is in fact being attacked by protease?
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