1. a . Lot of people do, so yes, and it is the only option if it is not a recombinant protein!

1.b. In ur case, no, because u have His tag.

2. Yes, U should use Mono Q

3. Here is ur answer.

Step 1: Make two buffers, (either Tirs/HEPES/Phosphate), one with 200-400 mM NaCl and other with 50 mM NaCl (both pH > 7.2).

Step 2: Lyse the bacteria in high salt buffer ( additionally 10 or 20 mM Imidaozle) and load on His column.

Step 3: Wash 10 c.v with high salt and 20 mM imidaole. Later, wash the column with 10 cv with low NaCl buffer and 20 mM Imidaole.

Step 4: Elute the protein with low NaCl buffer wit 250 or 300 mM Imidaole. For. Eample, Tris, Ph8.0, 50 mM NaCl and 300 mM Imidaole.

Step 5: Equilibriate the Q sepharose, with low salt buffer (Tris/HEPES / Phosphate 50 mM NaCl). Load ur eluted protein.

Step 6: Wash only 1 CV each with high NaCl buffer in increasing concentrations, 1cv 50 mM NaCl buffer, 1 CV 100 mM NaCl. etc.

for ur protein, with a PI of 5.8, I presume, it should elute at 300 mM Nacl.

So, typical eg buffer is Tris, pH 8.0, 300 mM NaCl. It should be very pure.

Feel free to ask me if u have any further doubts!!

thank you bhanu for the elaborate steps.

in the first case, where in the crude ISF is used, how am supposed to load it on the column (Mono Q) connected to NGC biorad system (fplc system)? I mean is the ISF prepared in the same manner or different steps are included to make it more enriched with the target protein (without any round of Affinity purification since IMDZ is involved in the elution buffer)?

It should be the same (though I have never tried), but make sure the lysis buffer has 50 mM NaCl, for proper binding on to the Q column. In long run, it may block the loops and/or the column. But if it is a resin and u r doing manually, u will not have many problems. Nevertheless the protein, will still not be 100 % pure. So, after monoQ, u still have to do FPLC or His affinity.

Hello Bhanu,

I also have a protein with a pi of 5.8. After a first purification with a HiTrap column I have a higher purity but not enough for my experiments, so I also thought about an anion exchange chromatography. So you only work with two buffers of different salt concentrations. But what pH values do you use for your chromatography. I don't understand the sixth step of the elution. Can you please help me to understand it better?

Cheers

Marco

Hello Marco,

Its quite simple. First thing, the pH should be constant in all cases, weather it is His-trap or anion exchange chromatography.

To describe step 6 in detail, I make two buffers for this. The first one is 20 mM buffer (Tris/HEPES/Phosphate etc and NO SALT). The other buffer is 20 mM buffer + 1 M NaCl. These buffers will have same pH, say 7.5 or 8 etc and its better to have same pH from step 1. Now, using simple M1.V1 = M2.V2 formula, make different concentrations of NaCl, for eg, to make 100 mM NaCl buffer (10 ml), take 1 ml of second buffer (with 1M NaCl) and add 9 ml of first buffer (without NaCl). This way make 200, 300 and 400 mM NaCl concentrations, and see where your protein is coming.

If your lab can afford Strep resin, change your tag to Strep resin and you will get ~ 100 % pure protein at the affinity level itself. Nevertheless, if you can optimize the way I mentioned above, you should certainly see an improvement in the purity.

Do let me know,if you still have queries.

Cheers

Bhanu