Unable to identify our protein - need an idea (Apr/19/2007 )
QUOTE (swanny @ Apr 19 2007, 10:15 PM)
If your band is a mix of IgG and your protein of interest, you might be able to take the IP fraction, dissociate the complex and purify by HPLC (ion exchange, reverse-phase might work best), then run the fractions on a gel and send off your multiple-purified protein
Sorry, I can't understand the situation deals with contamination with IgG fraction your protein, when you've made SDS-PAAG and identify your protein according its mass and cut only this band???
What is about Mw of your protein? Try to do it in non-reducing conditions and so Ig G band will be at the top of gel (near 150kDA) and you can separate each other
-circlepoint-
QUOTE (swanny @ Apr 19 2007, 10:15 PM)
If your band is a mix of IgG and your protein of interest, you might be able to take the IP fraction, dissociate the complex and purify by HPLC (ion exchange, reverse-phase might work best), then run the fractions on a gel and send off your multiple-purified protein
or FPLC Superose 12 or Superdex 200 gel filtration columns
-circlepoint-
QUOTE (circlepoint @ Apr 20 2007, 12:42 PM)
QUOTE (swanny @ Apr 19 2007, 10:15 PM)
If your band is a mix of IgG and your protein of interest, you might be able to take the IP fraction, dissociate the complex and purify by HPLC (ion exchange, reverse-phase might work best), then run the fractions on a gel and send off your multiple-purified protein
Sorry, I can't understand the situation deals with contamination with IgG fraction your protein, when you've made SDS-PAAG and identify your protein according its mass and cut only this band???
What is about Mw of your protein? Try to do it in non-reducing conditions and so Ig G band will be at the top of gel (near 150kDA) and you can separate each other
50 kDa, written in the 3. or 4. post.... and there is the heavy chain (denaturating conditions...)
-ms-olli-
QUOTE (circlepoint @ Apr 20 2007, 03:42 PM)
Sorry, I can't understand the situation deals with contamination with IgG fraction your protein, when you've made SDS-PAAG and identify your protein according its mass and cut only this band???
What is about Mw of your protein? Try to do it in non-reducing conditions and so Ig G band will be at the top of gel (near 150kDA) and you can separate each other
What is about Mw of your protein? Try to do it in non-reducing conditions and so Ig G band will be at the top of gel (near 150kDA) and you can separate each other
I did SDSPAGE, cut the band I saw (coomasie) at 50 KDa (which is the MW of my protein), sent it to MS and they reported me that there was IgG but no any other protein.
I though that denaturing conditions of the gel would have disrupted the Ab-antigen interactions, but it seems that they comigrate together and there is no way to identify my protein in that band.
If I do non-reducing conditions, I´ll maintain the union between Ab-antigen and they will migrate together, won´t they?
-Pumuki-
QUOTE (Ceri @ Apr 20 2007, 03:32 PM)
I haven't ever tried anything like this, but if you run 2 2D gels and transfer one to a Western membrane and identify your protein of interest with your monoclonal/polclonal ab. Then identify this protein on the other gel which is coomasie or sliver stained and cut it out for MS. Would that work?
Ceri
Ceri
hey, that seems easy! THANKS A LOT, ceri
I will try it, although serum has a lot of proteins and it could be a little confusing with so many spots. But perhaps if I use for first dimension a strip with a narrow pH range it may be easier.One guy in my lab once sent a silver stained spot from a 2D gel and the people from the MS service said it was not enough, they asked for a coomasie stained spot. Is it possible to identify a silver stained spot, is MS sensitive enough or do you always need coomasie? (sometimes there´s no enough sample and you cannot see your spot with coomasie)
-Pumuki-
QUOTE (Pumuki @ Apr 20 2007, 07:16 AM)
QUOTE (circlepoint @ Apr 20 2007, 03:42 PM)
Sorry, I can't understand the situation deals with contamination with IgG fraction your protein, when you've made SDS-PAAG and identify your protein according its mass and cut only this band???
What is about Mw of your protein? Try to do it in non-reducing conditions and so Ig G band will be at the top of gel (near 150kDA) and you can separate each other
What is about Mw of your protein? Try to do it in non-reducing conditions and so Ig G band will be at the top of gel (near 150kDA) and you can separate each other
I did SDSPAGE, cut the band I saw (coomasie) at 50 KDa (which is the MW of my protein), sent it to MS and they reported me that there was IgG but no any other protein.
I though that denaturing conditions of the gel would have disrupted the Ab-antigen interactions, but it seems that they comigrate together and there is no way to identify my protein in that band.
If I do non-reducing conditions, I´ll maintain the union between Ab-antigen and they will migrate together, won´t they?
Well! Sorry for Mw I was blind but I think boiling SDS in sample preparation without BME will be helpfull to dissociate the complex. Try to make one more PAAG
Good Luck!
-circlepoint-
[/quote]
Well! Sorry for Mw I was blind but I think boiling SDS in sample preparation without BME will be helpfull to dissociate the complex. Try to make one more PAAG
Good Luck!
[/quote]
thanks circlepoint, I´ll give that a try.
-Pumuki-
QUOTE (Pumuki @ Apr 20 2007, 02:31 PM)
QUOTE (Ceri @ Apr 20 2007, 03:32 PM)
I haven't ever tried anything like this, but if you run 2 2D gels and transfer one to a Western membrane and identify your protein of interest with your monoclonal/polclonal ab. Then identify this protein on the other gel which is coomasie or sliver stained and cut it out for MS. Would that work?
Ceri
Ceri
hey, that seems easy! THANKS A LOT, ceri
I will try it, although serum has a lot of proteins and it could be a little confusing with so many spots. But perhaps if I use for first dimension a strip with a narrow pH range it may be easier.One guy in my lab once sent a silver stained spot from a 2D gel and the people from the MS service said it was not enough, they asked for a coomasie stained spot. Is it possible to identify a silver stained spot, is MS sensitive enough or do you always need coomasie? (sometimes there´s no enough sample and you cannot see your spot with coomasie)
Thats always my problem, i´m the ms-man and all the people in our labs giving faint coomasie-bands to me
Silverstained spots could work ( !!! different protocols, only some did work with MS), exspecially with MALDI and peptide-mass-fingerprint. With ESI and MS/MS you have sometimes the challenge to get the sequence informations you need... AND i know the next problem. Start to identify the silver-spots and they comes the next day with faint silver-spots...
-ms-olli-
No prob 
EDIT: I don´t mind! I don´t feel offended, I understand the "here" right
-ms-olli-