what these components do in the buffer used in protein purification - (Nov/10/2006 )

Dear all.
just not really sure waht they do and what is the difference between in some very similar function staff for protein purificaition.

1. tween-20
2. tritonx-100
3. i know EDTA is a chelating reagent, break the metal cofactors for some protein, enzymes
what else?
3. Mgcl2
4. NP40
5.mercaptoethanol
6. by the way, NaCl and KCl what is the difference when use it in the buffer?

maybe stupid question, thanks all

---

5. is a reducing agent used to break sulfide bonds between AA

---

1 2 and 4 are detergents to solubilize membrane proteins.

---

3. EDTA chelates Ca which is necessary for enzymes, so it is a protease inhibitor

---

EDTA complexes Mg2+, Zn2+ and to a lower extent Ca2+ (EGTA is better for Ca2+), as it was said to inhibit metalloproteases; the reason for MgCl2 in your buffer is unclear to me; in the presence of EDTA you have a EDTA-Mg2+ buffer system; Mg2+ is used for ATP-dependent reaction and presumably to stabilize protein conformation; the same is true for Na+ and K+; normally, Na+ is in excess to K+ as cells were evolved to higher concentrations than K+, so preparation buffer contains more NaCl than KCl

---

Thanks all. that is really helpful. For the three detergent, are they different on level? eg, which one is more harsh? thanks

---