6 replies to this topic

[Negar]

Hi everyone,
I have a protein sample (5.2 ml) with the following ingredients:

Mannitol (170 mg)
Sodium Citrates (70 mg)
Polysorbate 80, NF (0.53 mg)
Citric acid/ NaOH (to adjust pH)

I want to electrospray the sample into a Mass spec. Just wondering what's the best method to get rid of salts, detergent, and acid/base from my sample? Thanks

[K.B.]

Desalting column eg. PD-10 from GE Lifesciences or Zebra from Pierce. (If you have Sephadex G-25 or similar gel and syringe barrel you can make a desalting column yourself.)

[mdfenko]

or you can dialyze against a more compatible buffer.

[Negar]

mdfenko, on 03 September 2010 - 08:17 PM, said:

or you can dialyze against a more compatible buffer.

Thank you all! I think dialysis against Ammonium acetate buffer should work...I dont know what concentration of buffer though?!! Also, dialysis is not gonna remove the detergent, is it?!

Edited by Negar, 03 September 2010 - 09:57 PM.

[Negar]

K.B., on 03 September 2010 - 11:55 AM, said:

Desalting column eg. PD-10 from GE Lifesciences or Zebra from Pierce. (If you have Sephadex G-25 or similar gel and syringe barrel you can make a desalting column yourself.)

Thanks a lot! I will try PD-10 columns next week, any tips on using them?

[Vini]

Negar, on 03 September 2010 - 09:58 PM, said:

K.B., on 03 September 2010 - 11:55 AM, said:

Desalting column eg. PD-10 from GE Lifesciences or Zebra from Pierce. (If you have Sephadex G-25 or similar gel and syringe barrel you can make a desalting column yourself.)

Thanks a lot! I will try PD-10 columns next week, any tips on using them?

just pass about 15 mL MQ, followed by 15 mL of the desired buffer through the PD-10 column. Then put 2.5 mL of your protein, collect the flowthrough separatly (in case you wanna check if the protein hasn't come out in this!). Then elute out your protein through the column with 3.5 mL of buffer. I think you can follow this up by concentrating your protein, before you put it into the mass spec. I guess, Ammonium acetate buffer would be a good choice, because it really helps the fragments fly in negative ion mode. Maybe you can use 20 mM buffer...hope this helps

Edited by Vini, 11 September 2010 - 10:49 PM.

[mdfenko]

Negar, on 03 September 2010 - 09:57 PM, said:

Thank you all! I think dialysis against Ammonium acetate buffer should work...I don't know what concentration of buffer though?!! Also, dialysis is not gonna remove the detergent, is it?!

extensive dialysis can be used to remove detergent (sds can be removed by dialysis against triton which can then be removed by dialysis against buffer).

ammonium acetate is a volatile buffer (for ms purposes) but should be limited to 10-20 mM.

Edited by mdfenko, 17 September 2010 - 08:27 AM.