It would be costly to use them for all steps. One vial of PI itself is more than 300$

Add all of them during lysis step then you can reduce concentration. Like, after lysis or during first step you can just use PMSF, and reduce PI upto 0.25 X, later for next step you can totally remove PI and only keep PMSF and you can also reduce PMSF conc at every step. PMSF itself is really good protease inhibitor.

If your protein is very unstable or if your lab dont care how much you spend then do whatever works for you.

Amended on 2 February 2019

This is an intrigueing, suffering, and harassig issue in the biochemistry.

I have luckily purified the human serum biotinidase (1-) from human blood without using any protease inhibitors and EDTA (please see file; Thiol-type BIN). But, this is a good fortune for us, since usual serum-constituent proteins such as Albumin is maintained their chemical structure intactly; i.e., Albumin can be purified from 25 – 609 of precursor, Transferrin is purified from 20-678 of precursor, Ceruloplasmin is purified from 19-1065 of precursor (therefore, I have found that Ceruloplasmin fragment (214-1065) is surely the marker of the inflammation (please see files; JMBT alopecia and The Fascio effect). This result of serum may also be due to rich in Alpha2-Macroglobulin/Alpha-2-M/A2M (24-1474 of precursor) and Alpha1-Antitrypsin/A1AT/A1A/Serpin (25-418 of precursor).

As a reference, I have calcuated the content of A2M and A1A among human serum (n = 13). Medians of A2M and A1A are 2.8 (range; 1.8 - 13.8 ) and 1.8 (range; 0.5 - 3.8) μg/mg of serum protein, respectively. Interestingly, highest value is of the serum of common cold patient.

On the other hand, non-constituent proteins in human serum have already been processed (please see file again; JMBT alopecia). Thus, we have previously purified the biotinated fragments derived from carboxylases and unkown biotin-containing proteins from human blood by using avidin- and/or streptavidin-affinity chromatography (please see file; BIN Peptide Inhibitor).

Therefore, addition of protease inhibitors during purification seems to be in vain. Further, we have found that biotinidase (thiol-type amidohydrolase) has turned out to have an amino-exo-peptidase activity to be called as Biotinidase/Enkephalin-exopeptidase (please see file; Enk serumBIN). Pig brain lipoamidase has been shown to be a multiple hydrolase (please see file; Multiple hydrolase LIP).

The point seems to choose the biological specimens which contain target protein or protein of interest (POI) richly.

Pig cerebrum biotinidase has been purified without PIs, but has been performed with 1 mM EDTA (please see file; BIN Brain).

Pig brain lipoamidase has been purified by adding 1 mM EDTA, except WGA-affinity separation is performed without PIs (please see file; Purify Brain Lipoamidase).

Thus, biotinidase and lipoamidase are unexpectedy major brain protein.

Further, I have searched for Protease inhibitors/PIs among my protein database (please see file; HepG2 fucoidan), and have found that cultured cells and cancer tissues seem to contain lower PIs.

Protease inhibitors/PIs are significantly lower in the Cancer tissues than the Normal tissues (p = 0.05, single-sided test; Mann-Whitney's U test n1 = 3, n2 = 3). Thus, protein purification from cultured cells and cancer tissues may be more difficult than normal organs.

Fetal hepatocyte Hc has surprisingly no Protease inhibitors/PIs.

Cancer tissues;

HCC tissue (with PBC) has Beta-Casein at 0.77 μg/mg of tissue protein. HCC tissue (named as No.6) has Alpha-2-Macroglobulin/Alpha-2-M/A2M at 0.19 μg/mg of tissue protein.

Hepatoma HepG2 (cultured without fucoidan) has Metalloproteinase inhibitor 4/Tissue inhibitor of metalloproteinases 4/TIMP-4 at 0.85 μg/mg of cell protein.

Normal tissues;

LC tissue (named as No.6) has Amyloid beta A4 protein/Alzheimer's disease amyloid A4 protein/PN-II at 0.25, Heparin cofactor II/HC-II at 1.7, and Plasma serine protease inhibitor/Plasminogen activator inhibitor 3/PAI-3 at 1.6 μg/mg of tissue protein, respectively. Total PIs becomes to be 3.55 μg/mg of tissue protein.

LC tissue (with leprosy) has Cystatin S at 0.49, Heparin cofactor II/HC-II/HLS2 at 1.3, Kallman syndrome protein/KAL1/Anosmin-1 at 3.9, and Metalloproteinase inhibitor 1/TIMP-1/EPA at 1.6, and Orosomucoid/Alpha-1 Acid glycoprotein 1 at 1.6 μg/mg of tissue protein, respectively. Total PIs becomes to be 8.89 μg/mg of tissue protein.

Normal liver (with pseudo-liver cancer) has Alpha-2-Antiplasmin/Alpha-2-PI/A2AP at 1.2, and Alpha-2-Macroglobulin/Alpha-2-M/A2M at 0.98 μg/mg of tissue protein, respectively. Total PIs becomes to be 2.18 μg/mg of tissue protein.

Hi there,

Usually you can avoid protease inhibitors as soon as you are in the purification process. Except if protease contaminant is copurifying along with your POI...

Dear Aron

i'm agree with Dominique.

Generally the protease inhibitors are usefull to protect proteins from degradation beetween lysis and purification, when the protein is in contact with the proteases present in the lysate and therefore you can add it in the LYsis/binding buffer.

Once the protein is purified is less probable that protease remain with it, generally is enough to store the sample at 4°C.

good luck

Manuele

PMSF is not in inhibitor of proteases, but an inactivator. Once it has reacted with the protease, it will not go away again. Therefore, it is sufficient to have it during lysis. In fact, as the half-life of PMSF in water is only ~20 min, there is little point to add it to, say, chromatographic buffers.

True inhibitors like benzamidine or leupeptin, which are in a dissociation-association equilibrium with the proteases, need to be present as long as the protease is present.

Article Inactivation of the protease inhibitor Phenylmethylsulfonyl ...

https://www.thermofisher.com/order/catalog/product/36978

For more information attached is paper, and overview of pmsf - It is an excellent choice. To add pmsf - I always pick up fresh vial right before every step of purification, and it helps. Whatever works -Its your call - Good luck

Thank you all for the reply!

I agree that adding them in the lysis buffer should work and enough but I had an issue with degraded protein fragments during the Flow through of NiNTA binding and subsequent washings, So either I should increase the PMSF/PI?Pepstat concentration during the Lysis buffer so that it can be more effective or there are some other reasons to see the cleavage of POI even though adding them during lysis?

To add to Engelbert Buxbaum’s answer, PMSF reacts with serine proteases, forming a covalent bond so you do not have to add it to subsequent buffers. The same goes for AEBSF that also inhibits serine protease and also for E64, used to inhibit some cysteine proteases. Pepstatin, which you mention in your question, is an example of an inhibitor that binds reversibly so, when absent in the buffer, some of the pepstatin that is inhibiting the protease comes free, leaving the protease in active form. However, pepstatin is rather expensive to be adding to large volumes of buffer. So, I go by the pH of the buffer. Pepstatin is usually added to inhibit acidic proteases (the classical example is pepsin) which have optimum pH in the acidic range, so I would leave it out when the buffer is at alkaline pH. Another common inhibitor added is EDTA to inhibit metalloproteases. I would add EDTA to the buffers.

Sigma sales his compatible protease inhibitors you can use 2X of that stuff during lysis, you should also add metal chelators during lysis, however you should avoid them during his purification. You can add 2X PI, PMSF, EDTA, and also individual PI during lysis and see if it helps. If you have degradation then your protein is quite unstable, you will have to consider if you can optimize and find stabilizing conditions ( pH, buffer, ligands, lipids etc) and protease inhibitor is only one of the condition.

Agree with Mahesh above - your protein seems to be unstable and this may not be due to proteases (but yes, PIs in just the lysis buffer as everyone has said). DTT and EDTA in your storage buffer helps, but your degradation is 'on column'. If you are being very careful keeping everything cold during all your steps (esp. during sonication) then you may need to consider re-cloning. You said 'Ni-NTA column' so is your Hisx6 tag C- or N-term? If it is N-term then your degradation is happening at the C-term, so losing some of the C-terminus (eg up to the active domains of your POI) may translate to a more stable protein. Or switch tags to the other end, degradation products will be washed away and add DTT/EDTA to your combined eluted fractions etc then dialyse with these present as well. Increase your NaCl to 500mM (all steps) if you haven't already (up to 1M even) might help also. re: HEPES and MOPS were my 'go-to' buffers but as Mehesh says, your protein may like slightly more acidic or basic conditions...? Good luck

Thanks All,

I did try 2X and it was working a little better but not changed life much.

Adding EDTA can cause problem of NiNTA it will remove NiNTA, I may try minimum concentration but not sure if it will cause any reduction with the POI binding to NiNTA.

The His Tag is on C terminal and I need to elute protein at RT. I have N terminal MBP tag but the problem is that binding to MBP is also not much good. High salt making it more difficult. I use 500mM in the NiNTA elution buffer which I use for the MBP binding incubating on RT for 2-3 hrs with gentle shaking.