1. You can use a general inhibitor of serine proteinases such as PMSF (phenylmethane sulphonylfluoride). If you do, then take precautions: make it up fresh before use in, in an organic solvent such as acetone (because it will happily react with water if there's no trypsin) and make sure that you have added enough to really kill the trypsin. Even a little bit of residual proteolytic activity can chew up your peptides once a protein is unfolded.

2. If you want to be more specific and expensive you can use TLCK which is selective for trypsin.

3. A good step, whatever else you do, is simply to terminate reaction by taking the pH a long way from where trypsin is active - e.g. pH 4.

4. Finally, not a bad idea to actually test for residual activity by having a solution of a synthetic chromogenic trypsin substrate like BAPNA.

HPLC, SDS-Page, Gel Filtration

Use Agarose immobilized trypsin. This can be removed by filtration.

We used sulphuric acid to stop enzymatic reactions. You probably try to stop protein cleavage, so you can use acetic acid. All the ways to move tripsine to low pH are useable. I think better way than by the heat denaturation.

You can stop the digestion with formic acid to 100%, 0.75 µl, and then frozen until analysis

A HiTrap benzamidine column (GE Healthcare) works well, but I recommend adding PMSF to 1 mM afterwards to irreversibly inhibit any trypsin that might have crept through.

You can stop the reaction with low pH and then keep froozen your samples. Usually you need to purify the products, in that case gel filtration works fine.

First, use heat denaturation to stop trypsin activity, then follow up with a suitable liquid chromatographic technique (eg, Ion-exchange or Gel filtration) to remove trypsin from the mixture. Peptides from your purified protein can be purified by using reversed-phase chromatography. You can use SDS-PAGE to track removal of trypsin and/or purification of peptides.

Hi Andrew!

I never did it myself but what you can use is tagged trypsin. For example you could use trypsin that is fused to magnetic beads. After digestion you can easily remove the trypsin using the beads. Or take any other tag that you like.

You can get the magentic bead labeled trypsin from clontech (http://www.clontech.com/US/Products/Protein_Expression_and_Purification/Reagents/TPCK_Trypsin)

1. You can use a general inhibitor of serine proteinases such as PMSF (phenylmethane sulphonylfluoride). If you do, then take precautions: make it up fresh before use in, in an organic solvent such as acetone (because it will happily react with water if there's no trypsin) and make sure that you have added enough to really kill the trypsin. Even a little bit of residual proteolytic activity can chew up your peptides once a protein is unfolded.

2. If you want to be more specific and expensive you can use TLCK which is selective for trypsin.

3. A good step, whatever else you do, is simply to terminate reaction by taking the pH a long way from where trypsin is active - e.g. pH 4.

4. Finally, not a bad idea to actually test for residual activity by having a solution of a synthetic chromogenic trypsin substrate like BAPNA.

try to inactivate it by heat (55-60 C).

Dear Sir,

You may use immobilized trypsin or immobilized trypsin inhibitor to remove trypsin after reaction (you'll find it in the literature).

Hi everyone, thank you for the great (and rapid) information. The suggestions are all great and high quality (as per usual), I am keen to hear more but the immobilised trypsin is a my favourite suggestion so far and could possibly fit well with the application I have in mind.

I knew you wouldn't let me down.

Cheers,

Andy

If the fragments are of lower molecular weight and small reaction volume is to use a quick solution microcentrifuge filters (NMWL: 10.000). Other solutions is inject the sample into an HPLC and separate them by gel filtration. Each fragment will have a Tr (retention time characteristic) and individually if you can separate each fragment if you wanted y and obviously you separate trysina signal having a molecular weight clse 23 kDa

Immobilised Benzamide capture of Trypin and PMSF addition as a precaution is good for existing maintaining the existing prior trypsin treatment conditions. Immobilised trypin may require preliminary optimisation if you have pre-existing free trypsin conditions. Either way, using DL BApNA reaction rates can identify free-removed trypsin and/or adjust immobilised trypsin to free trypsin hydrolysis conditions.

Not a fan of kits, so make a tonne of a trypsin affinity column yourself using 4-amino benzamidine and CNBr activated Sepharose, for example. Works a charm - was using this in 1978!

You can also use immobilised soybean trypsin inhibitor or lima bean inhibitor-again, easy to make, but also available commercially.

A word of caution - in the previous millenium I was looking at limited proteolysis of native proteins. I had assumed that boiling in SDS would kill the protease. But, there were no limited proteolysis products. However, when I inhibited the trypsin first, bands were there. I transpired that in the minute or so of heating in SDS, the substrate protein denatured easily, but trypsin, being mostly beta sheet, tok a little longer, and in its death throes, went to town on my protein and completely digested it. I would not rely on all denaturants to kill trypsin quickly enough.

I would go for TLCK - it is very smelly (it smells of mice, to me!) but is fast and effective. And, it is irreversible. PMSF - too many hassles over toxicity and instability. Then, use a small amount of immobilised inhibitor to remove the trypsin (pass the digest over a small volume of beads).

All of these methods were really well know in the 1980's, but seem to have fallen by the way now. I recommended a trip to the place where PDFs still exist in paper form, and look up Methods in Enzymology or Advances in Protein Chemistry :) Or even, go and see if your library has a copy of Proteolytic Enzymes: A Practical Approach - sorry for the advert, but the book is probably remaindered, although it still contains a lot of good, timeless advice. I don't recommend buying it! Asking me for advice is cheaper. Rob

http://books.google.co.uk/books/about/Proteolytic_Enzymes.html?id=3ddVeNO7tmgC&redir_esc=y

Simple answer is soybean trypsin inhibitor from Sigma.