Protein purification with HPLC - (Jul/23/2007 )
Hi
I need to purify my protein further (probably with a MONO Q or MONO S column) but I've never used a HPLC before . Does anyone know the protocol with these columns? ie. wash with water or ethanol (number of times or volume needed) and what types of buffer I need to make (my current protein is in PBS buffer). Thanks
I need to purify my protein further (probably with a MONO Q or MONO S column) but I've never used a HPLC before . Does anyone know the protocol with these columns? ie. wash with water or ethanol (number of times or volume needed) and what types of buffer I need to make (my current protein is in PBS buffer). Thanks
you need two buffers:
A for loading and washing and building a gradient with B during elution
good buffers to start with are
A: 40 mM Tris/HCL pH 7.4 (optional 0.5 mM DTT)
B: 40 mM Tris/HCL pH 7.4 (optional 0.5 mM DTT), 1 M NaCl
wash with 10 volumes of column, elute with 1-2 volumes of column in a steep gradient
Thanks... btw, do you know if you can use PBS buffer and just add some salt?
I need to purify my protein further (probably with a MONO Q or MONO S column) but I've never used a HPLC before . Does anyone know the protocol with these columns? ie. wash with water or ethanol (number of times or volume needed) and what types of buffer I need to make (my current protein is in PBS buffer). Thanks
you need two buffers:
A for loading and washing and building a gradient with B during elution
good buffers to start with are
A: 40 mM Tris/HCL pH 7.4 (optional 0.5 mM DTT)
B: 40 mM Tris/HCL pH 7.4 (optional 0.5 mM DTT), 1 M NaCl
wash with 10 volumes of column, elute with 1-2 volumes of column in a steep gradient
Keep in mind the pI of your protein, if you know it. The pH of your buffers and the pI of your protein will affect whether your protein will bind to the column, or whether it will flow through. Until you know how your protein behaves on the column with the buffers (and pH you choose to work with), be sure to collect everything that comes off the column: flow through, wash and all fractions. What you want to avoid is a buffer-column combination that allows your protein to bind weakly (you'll get some protein in the flow through (not bound) and some eluted in your fractions. If this happens, adjust pH or change columns.
I usually use MES buffers at pH 6.5 for the Mono S column and Tris-HCl at pH 8 for the Mono Q. Again, your protein can behave quite differently on each column or with each pH...
Remember: if the pH of the buffer is higher than the pI of the protein, the protein will be negative and thus will bind more tightly to a positively charged column. Conversely, if the pH of the buffer is lower than the pI of the protein, the protein will be positivle charged and bind more tightly to a negatively charged column.
I hope I didn't confuse you.
Good luck!
I usually use MES buffers at pH 6.5 for the Mono S column and Tris-HCl at pH 8 for the Mono Q. Again, your protein can behave quite differently on each column or with each pH...
Remember: if the pH of the buffer is higher than the pI of the protein, the protein will be negative and thus will bind more tightly to a positively charged column. Conversely, if the pH of the buffer is lower than the pI of the protein, the protein will be positivle charged and bind more tightly to a negatively charged column.
I hope I didn't confuse you.
Good luck!
Some additions to tercli07 reply: It will be better if you know pI of your protein prepare buffer solution with pH near 1 point higher or lower of pI. So you will elute your protein in middle range of salt gradient ( near 150-200mM NaCl). If you choose pH far from pI so you protein will bind stronger to column and you will need high salt concentration to elute protein. Why is it important? first of all some proteins don't like high salt concentration and precipitation will occur during purification. The other very important - HPLC system I mean plungers in pumps and stainless steel tubing ( if you have PEEK tubing like on HP Agilent systemsthis problem decreases but not for pump heads) are very sensitive for NaCl and corrosy damage will occur. Also from my experience try to minimise use of buffers which contain more than 30mM phosphate in composition, because phosphates are susceptible for temperature depended precipitatation in HPLC tubings. Of course it is possible to minimise or get rid of these problems if after purification you immediately wash your system with deionised water ( just put two ends of tubings( from A and B ways ) in one bottle with water and wash near 30-60 min. Also if you have automatic plunger wash system on your device it will be good for long-life of your pumps when you are working on IEC HPLC.
Adittion to common buffers used in protein isolation IEC: Na citrate( 20mM pH range 2.6-3.6) Na acetate 50mM pH range 4.8-5.2; HEPES 50mM ( pH 7,6-8.2) Na phoshate 20mM ( pH 7,2) ethanolamine ( 20mM pH range 9-9.5) piperazine ( 20mM pH range 9.5-9.8 also available as buffer system at pH 5-6 ) other buffers I don't list because exotic and rare used in common IEC HPLC procedures.
Hope it helps!
Hi Everyone
The pI of my protein is 6.6 and I want to use a MONO S column. Does that mean I should make my MES buffer at around pH 5.6? Also my protein is now in PBS with 150 mM salt + 0.1%. Do I need to buffer exchange into a lower salt buffer before adding to MONO S column (coz for mono s column , proteins already starts eluting at 200 mM???)
Also, I was going to make my start buffer 20 mM MES, PH 5.6
and my elution buffer 20 mM MES + 1.0 NaCl, pH 5.6
but if want it to elute at middle range, do i need to make multiple buffers with various salt concentrations?
Thanks so much
I usually use MES buffers at pH 6.5 for the Mono S column and Tris-HCl at pH 8 for the Mono Q. Again, your protein can behave quite differently on each column or with each pH...
Remember: if the pH of the buffer is higher than the pI of the protein, the protein will be negative and thus will bind more tightly to a positively charged column. Conversely, if the pH of the buffer is lower than the pI of the protein, the protein will be positivle charged and bind more tightly to a negatively charged column.
I hope I didn't confuse you.
Good luck!
Some additions to tercli07 reply: It will be better if you know pI of your protein prepare buffer solution with pH near 1 point higher or lower of pI. So you will elute your protein in middle range of salt gradient ( near 150-200mM NaCl). If you choose pH far from pI so you protein will bind stronger to column and you will need high salt concentration to elute protein. Why is it important? first of all some proteins don't like high salt concentration and precipitation will occur during purification. The other very important - HPLC system I mean plungers in pumps and stainless steel tubing ( if you have PEEK tubing like on HP Agilent systemsthis problem decreases but not for pump heads) are very sensitive for NaCl and corrosy damage will occur. Also from my experience try to minimise use of buffers which contain more than 30mM phosphate in composition, because phosphates are susceptible for temperature depended precipitatation in HPLC tubings. Of course it is possible to minimise or get rid of these problems if after purification you immediately wash your system with deionised water ( just put two ends of tubings( from A and B ways ) in one bottle with water and wash near 30-60 min. Also if you have automatic plunger wash system on your device it will be good for long-life of your pumps when you are working on IEC HPLC.
Adittion to common buffers used in protein isolation IEC: Na citrate( 20mM pH range 2.6-3.6) Na acetate 50mM pH range 4.8-5.2; HEPES 50mM ( pH 7,6-8.2) Na phoshate 20mM ( pH 7,2) ethanolamine ( 20mM pH range 9-9.5) piperazine ( 20mM pH range 9.5-9.8 also available as buffer system at pH 5-6 ) other buffers I don't list because exotic and rare used in common IEC HPLC procedures.
Hope it helps!
Well you are right! You should use your buffer at 5.6. ( MES is expensive so you could use acetate buffer at pH 5.5 ) / Before IEC you must dialyse your protein against starting buffer ( MES or 50mM acetate without salt). Buffer exchange you may do on Sephadex G-25 also. IT IS A RULE! If you will use PBS so your protein partially fall through the column during appling on column.
"Also, I was going to make my start buffer 20 mM MES, PH 5.6
and my elution buffer 20 mM MES + 1.0 NaCl, pH 5.6" - right.
Decreasing in buffer pH in your case will increase salt concentration for elution of your protein!