7 replies to this topic

[cardosopedro]

Hello.

I work with a model organism and based on my results I have some candidates proteins I believe are new Ca2+-binding molecules. How can I prove this? I don't have the recombinant protein (and I don't intend to produce them) but I have KO mutant cells for these genes/proteins. How could I prove, directly or indirectly, that these are in fact new Ca2+-related proteins?

So far I did growth analysis on culture medium with different concentrations of Ca2+ and these mutant cells behave differently from the wild type. I also transformed these cells with a genetic reporter that allows intracellular Ca2+ measurement.

Please help.


Thanks a lot!!!

[mdfenko]

have you isolated these proteins?

if so then you can sequence them on lc/ms/ms to see if they have been identified.

if not then isolate and sequence them (you can separate them on a gel (2d, if necessary), cut them out and sequence them).

Edited by mdfenko, 10 May 2013 - 03:54 AM.

[cardosopedro]

The genome of the organism I work with is fully sequenced so I already have the sequence of these proteins. I predicted the presence of Ca2+-binding motifs using this tool (http://gallium.gsu.edu:8080/seq_f/) but I would to prove it biologically. At least indirectly, since I don't intend to produce the recombinant proteins.

[mdfenko]

since you have the wild-type and knock-outs you can compare them on a gel and stain for calcium binding proteins with stains-all.

you can get the procedure from this paper:

"staining of ca2+-binding proteins ... with ... stains-all"

[cardosopedro]

Thank you. I'll definitively try that.

[cardosopedro]

I was reading the paper you sent me and also other literature about this stains-all reagent and I although I'm leaning to try it, I was wondering about this: the idea is to compare stains all-stained SDS-PAGE gels loaded with protein extracts from the wild type vs the knockouts for my candidate proteins, right? However, since each extract contains thousands of proteins, that is, lots of bands in the gel, I was wondering if my eye will be sensitive enough to detect the lack of just one band in the lanes with the extracts from the knockouts...

Apparently, from what read, people normally use stains-all to stain purified protein rather than extracts. In that paper you recommended they use extracts but from sarcoplasmic reticulum vesicles which means they have a restricted number of proteins in the gel...

Please give me your opinions.

Thanks!

[mdfenko]

when i tried it (a long time ago), i used a partially purified protein (it had been through ammonium sulfate fractionation and 2 chromatography steps, if i remember correctly, or it may have been samples from the entire purification scheme) and it worked (i was thrilled).

i knew which band i was looking for but i was also looking to see if there was any other ca binding proteins present (if i remember correctly, there were some).

if there is a high enough concentration of your protein of interest then it should stain in the wild type sample. you may be able to identify the protein if you analyze an image of the gel. it will also help if you know approximately where to look on the gel.

(it is not a given that my recollection is correct)

Edited by mdfenko, 14 May 2013 - 04:34 AM.

[cardosopedro]

I guess I'll try it and see what happens.