3 replies to this topic

[efendy]

Hello from down under!

I am a PhD candidate at one of the Australian unis. I always found protocol-online very helpful throughout my studies but didn't have a specific question till today

here it comes: i was making an elution buffer for purifying my his-tagged protein and i added my 500mM S.phosphate stock (50mM), NaCl for 300mM and made it upto 800ml then checked pH. pH was 7.0 as I wanted, then added enough imidazole for 500mM (sigma - reagent plus - 99% - I202-100G) and made it upto 1000ml and checked pH. it elevated to pH 9.0! at this situation what do you do? i adjusted the pH using 32% HCl (10M!) but it added so much Cl to bring the salt strength to almost 500mM (just guessing). so how do you prepare yours without affecting the amount of Cl?

Cheers

[parado]

efendy, on Jun 10 2010, 08:54 PM, said:

Hello from down under!

I am a PhD candidate at one of the Australian unis. I always found protocol-online very helpful throughout my studies but didn't have a specific question till today

here it comes: i was making an elution buffer for purifying my his-tagged protein and i added my 500mM S.phosphate stock (50mM), NaCl for 300mM and made it upto 800ml then checked pH. pH was 7.0 as I wanted, then added enough imidazole for 500mM (sigma - reagent plus - 99% - I202-100G) and made it upto 1000ml and checked pH. it elevated to pH 9.0! at this situation what do you do? i adjusted the pH using 32% HCl (10M!) but it added so much Cl to bring the salt strength to almost 500mM (just guessing). so how do you prepare yours without affecting the amount of Cl?

Cheers

i never use stock solutions when prepare elution buffer , just weight and mix every reagent  and the subsequent solution is almost at the PH you need.

[HomeBrew]

Your problem might be that you used just sodium phosphate, rather than a mix of monobasic sodium phosphate and dibasic sodium phosphate?  You need the mix of the monobasic (NaH2PO4·2H2O for the dihydrate) and the dibasic (Na2HPO4·7H2O for the heptahydrate) forms for it to be a buffer, and thus minimize pH changes.

[mdfenko]

HomeBrew, on Jun 15 2010, 07:44 AM, said:

Your problem might be that you used just sodium phosphate, rather than a mix of monobasic sodium phosphate and dibasic sodium phosphate?  You need the mix of the monobasic (NaH2PO4·2H2O for the dihydrate) and the dibasic (Na2HPO4·7H2O for the heptahydrate) forms for it to be a buffer, and thus minimize pH changes.

i have to disagree with you. phosphate is a buffer, mono and dibasic mix makes it easier to prepare a buffer of desired pH but they are not necessary. you can prepare a buffer using phosphoric acid and sodium (or potassium) hydroxide.

however, i read it that efendy used a stock phosphate buffer to prepare his solution. so the point is moot.

imidazole is also a buffer and will influence the pH of any solution to which it is added, especially in high concentrations. you can either preadjust the imidazole or make the adjustment before making to final volume. either way you will increase the amount of chlorine in the solution (assuming you use hcl), but this should not have any serious side effects.