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help with his tagged protein purification

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#1 pgrover



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Posted 01 April 2011 - 12:56 PM

Hi everyone,

I work with two proteins that have a pI of 7.8(A) and 7.1 (B) respectively, they are both ~26kDa each and are his-tagged.

I started purifying protein A (pI 7.8) using the GE healthcare hitrap column, charged with Ni ions. I use the following buffers:
Binding buffer: 20mM tris, 0.5M NaCl, 20mM imidazole, 5mM BME, 10% glycerol, pH 8.3
Wash buffer: 20mM tris, 20mM imidazole, 5mM BME, 10% glycerol, pH 8.3
Elution buffer: 20mM tris, 1M imidazole, 5mM BME, 10% glycerol, pH 8.3
I got nice and clean bands from the his column, and then put it on GF column to dilute imidazole out and further purify the protein. I used the following buffer:20mM tris, 100mM NaCl, 3mM DTT, pH 8.3
I got a very broad peak from that and the protein was eluted in ~200mL buffer. I also lost a large amount of protein at this step.
So, next I decided to dialyze the protein into dialysis buffer (20mM tris, 100mM NaCl, 3mM DTT, pH 8.3). Almost all the protein precipitated out of solution (more than 90%).For dialysis, I did 1hr, o/n, 1 hr at 4C.
After I purify this protein, I need to label it with a fluorophore. The optimum conditions for this are pH 6.5-7.5 in a phosphate based buffer.

I started purifying protein B (pI 7.1) using the GE healthcare hitrap column, charged with Ni ions. I used the same buffers as described above.
Again, I got nice and clean bands from the his column, and then dialyzed it under different conditions.
a. 20mM tris, 100mM NaCl, 3mM DTT, pH 8.3
b. 20mM tris, 200mM NaCl, 3mM DTT, pH 8.3
c. 20mM tris, 500mM NaCl, 3mM DTT, pH 8.3
d. 0.1M phosphate, 100mM NaCl, 3mM DTT pH 6.5
e. 0.1M phosphate, 200mM NaCl, 3mM DTT pH 6.5
f. 0.1M phosphate, 500mM NaCl, 3mM DTT pH 6.5
For tris based buffers, I did not get any precipitation.
For phosphate based buffers, I again got a lot of precipitation and lost ~90% protein.

So, I think that the proteins are precipitating because of the pH range. I am in a difficult situation because all pH values are in the same range.
When I move from pH 8.3 to 6.5, the proteins precipitate at their pIs (7.1 and 7.8) But I need the final pH in the range of 6.5-7.5
So, now I have decided to use a phosphate based buffer for the his purification at pH 6.5. When I searched literature, I found that the optimum pH for these columns is 7-8. Some people suggest using low pH for eluting the protein (instead of imidazole). Do you think I'll be fine if I change the pH for all buffers to 6.5 (phosphate based) and continue everything else the same way?
Also, do you have any other suggestions or comments regarding this?

Please reply at the earliest, thanks for your help!

#2 protolder



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Posted 03 April 2011 - 09:53 PM

Hola I donīt see any problem in running the nickel colum at ph 6.5 from the begining and making your extracts at this pH with phosphate and tthe other components 20mM imidazole, NaCl 0.5M , reducer and glycerol.In the quiagen information says thatHis have a pKa of 6.0 and under this value starts to be protonated, so doesnīt bound to the nickel ions, for that pH dropwise is used for elution, monomers elute at 5.9 and multimers at 4.5. Buena suerte

#3 GxK



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Posted 04 April 2017 - 05:47 AM



I am in a similar kind of situation where the pI of my protein is around pH 8.1. I tried to do purification at pH 8 and 7.6 but ended up with partial binding. I am just curious to know what happened when you tried to do the purification at pH 6.5



#4 labtastic



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Posted 04 April 2017 - 01:16 PM

Some things to consider:


For protein A, you say you got a large broad peak on GF. Did you concentrate your protein to <2% of the bed volume of your column? If not, then this is why your protein peak was so broad. If you did, the peak is broad because your protein is polydisperse (mixture of oligomers and aggregates). In this case, your protein will not be suitable nor reliable for any downstream fluorescence work.


For protein B, how do you know your protein is precipitating when it reaches it's pI and not because it's at pH 6.5?


You should also try desalting your proteins instead of dialysis. Takes a fraction of the time and buffer. GE makes small PD-10 and Nap5 columns for small volumes (0.5-2.5 ml). You can buy larger columns for FPLC's or make your own with G25 sephadex.  You can then test desalt small amounts into pH 6.5, 7, 7.5 and 8.3 to see what the cause of precipitation is. Frankly dialysis is a waste of time and reagents.


You can also try keeping the glycerol around during your buffer exchanges.


Here's what I would try if possible: express your protein with a SUMO-tag. This will almost certainly increase the solubility of your protein. Conveniently SUMO tags have no cysteines (I'm assuming your doing maleimide chemistry for fluorophore labeling). Purify at pH 7-7.5 with SUMO tag, desalt into same pH low salt buffer, label with fluorophore and quench unreacted fluorophore with thiol of your choice. Desalt mixture into pH 8.3 where it's stable (which also gets rid of excess dye/quencher), cleave off SUMO tag with SUMO protease, run mixture over nickel to remove SUMO tag and protease and the flow through will be what you need with no extra unwanted tags or residues.


I often see people fight over and over with their protein to get it stable/happy when most of the time it can be fixed by altering expression construct or ortholog. They do this because they hate cloning. Cloning is your friend. Being fast and precise with your cloning will make you uber protein chemist while everyone else is still fighting poorly behaved proteins.


Edit...oops didn't realize OP was from 2011!!  blink.png

Edited by labtastic, 04 April 2017 - 01:17 PM.

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