5 replies to this topic

[mehdiimani]

hi i am searching phosphorylation test for isoforms of my protein (Calprotectin from neutrophil cells) but i have not been successed.
happy new year
scincerely

Edited by mehdiimani, 26 December 2004 - 10:40 PM.

[Oleksii]

Hi,

I am looking for simple protocol to check phosphorylated state of a protein too. Up to this moment I am using metabolic labeling with 32P and 33P, but I found protocol which possible could be better. It is simply staining  of SDS gel with phosphospecific dye. The name of the dye is  Pro-Q and it is Invitrogen/Molecular Probes product. You can go to www.invitrogen.com, select Molecular Probes products and search for Pro-Q. I have not tried  it but I am going to do it.  Reference for this methods: Electrophoresis 2004, 25, 2526-2532. Maybe it helps you.

Alexei

[Oleksii]

One more possibility… If you have good antibody against your protein, you can immunoprecipitate it and than blot with ether anti-phosphoserine, anti-phosphotyrosine or anti-phosphothreonine. It is may work or not work for your protein.

Good luck!

Alexei

[Konrad]

Greetings,
I'm a paediatric gastroenterologist and starting PhD research into inflammatory states affecting children and a specific interest into Calprotectin and its application as a marker of inflammation in the gut (inflammatory bowel disease, gut permeability, food allergies, etc.).

'Purely' medical journals explain basic information on the validity of this molecule as an aid to test the presence or absence of disease in the gut.

However since I am a clinician, it is rather hard to find comprehensive information about this molecule's structure, its origins, its specific inter/actions and effects. I would be very grateful indeed if someone could point me in the right direction. Non-clinical literature searches have yielded alot of very detailed information that is confusing at best! Does a map of pathway/s exist? Is there just one chemical name for this molecule or do more exist?

I would be very happy indeed to read from anyone who could help me in this endeavour.

Many thanks,

Konrad

[mabusheh]

mehdiimani, on Dec 26 2004, 10:50 PM, said:

hi i am searching phosphorylation test for isoforms of my protein (Calprotectin from neutrophil cells) but i have not been successed.
happy new year
scincerely

hi
I am working with a phospho isoforms of a protein of 30Kda too.
to detect phosphoisoforms of the protein u need to immunoprecipitate it, then run on native PAGE. and immuno blot it with a mono or polyclonal antibody.
the phospho protein stain Pro Q diamond from invitrogen is not very specific, i tried it many times.
after running on the gel, u can stain the gel with sypro stain (biorad) then excise the band of interest compared to ur western results, then do a mass spec on it for phospho pepetide mapping
let me if it works

good luck

[mabusheh]

Oleksii, on Dec 27 2004, 11:25 AM, said:

Hi,

I am looking for simple protocol to check phosphorylated state of a protein too. Up to this moment I am using metabolic labeling with 32P and 33P, but I found protocol which possible could be better. It is simply staining  of SDS gel with phosphospecific dye. The name of the dye is  Pro-Q and it is Invitrogen/Molecular Probes product. You can go to www.invitrogen.com, select Molecular Probes products and search for Pro-Q. I have not tried  it but I am going to do it.  Reference for this methods: Electrophoresis 2004, 25, 2526-2532. Maybe it helps you.

Alexei

let me know if this dye works for you please
I tried many many times, with no luck, its not very specific