Hi everyone,
I expressed a protein (which is supposed a calmodulin binding protein) in pET23a, BL21 strain and I want to do binding assay with calmodulin just to show that my protein is a calmodulin binding protein. Does anyone know how to do? Any suggestion is appreciated.
Thanks
Philip
0
1 reply to this topic
#2
kfj
-
- Members
-
- 2 posts
member
0
Neutral
Neutral
Posted 14 November 2010 - 11:39 AM
Hi Philip,
I just came across this topic while looking for my own troubleshooting. It is a bit old topic and you might have already gotten good answers. I did quite a lot of Calmodulin binding assays long long time ago because my master's thesis was purification of PP2B. Anyway,what I did was...
( I am assuming you have a lot of purified calmodulin. if not, it is quite easy to do it, and I can probably find a good protocol).
1. couple Calmodulin with CNBr-activated sepharose beads to make Calmodulin Sepharose affinity column.
2. add Calmodulin binding protein to the beads in the presence of CaCl2 (0.1-1mM?).
3. wash the beads with Ca-containing buffer and add buffers containing tons of EDTA (or EGTA if something like Mg2+ is required...5-10mM?).
Calmodulin binding protein will be associated with the beads in the presence of Ca, but eluted when Ca2+ is removed.
In your case, since you have His-tagged proteins to test, I would assume you could do the other way around. Like...
1. prepare your HIs-tag protein attached to the Ni-Beads.
2. add calmodulin in in the presence of Ca or EGTA (EDTA would dissociate your protein from the beads). Analyze the presence of Calmodulin in teh flow-through fraction (by WB or CBB staining).
3. wash with Ca-containing buffer, and elute Calmodulin with EGTA, and analyze the fraction by WB or CBB.
I just came across this topic while looking for my own troubleshooting. It is a bit old topic and you might have already gotten good answers. I did quite a lot of Calmodulin binding assays long long time ago because my master's thesis was purification of PP2B. Anyway,what I did was...
( I am assuming you have a lot of purified calmodulin. if not, it is quite easy to do it, and I can probably find a good protocol).
1. couple Calmodulin with CNBr-activated sepharose beads to make Calmodulin Sepharose affinity column.
2. add Calmodulin binding protein to the beads in the presence of CaCl2 (0.1-1mM?).
3. wash the beads with Ca-containing buffer and add buffers containing tons of EDTA (or EGTA if something like Mg2+ is required...5-10mM?).
Calmodulin binding protein will be associated with the beads in the presence of Ca, but eluted when Ca2+ is removed.
In your case, since you have His-tagged proteins to test, I would assume you could do the other way around. Like...
1. prepare your HIs-tag protein attached to the Ni-Beads.
2. add calmodulin in in the presence of Ca or EGTA (EDTA would dissociate your protein from the beads). Analyze the presence of Calmodulin in teh flow-through fraction (by WB or CBB staining).
3. wash with Ca-containing buffer, and elute Calmodulin with EGTA, and analyze the fraction by WB or CBB.
