mass spectrometry analysis protein identification drive me crazy - (Jul/10/2007 )
Hi dear all,
I try to identify the protein complex by TAP tag. and all seems fine, I see big fat band of my bait protein by silver stain and confirmed by western blot. Then my nightmare started, I spend five months now to try to get MS to identify my protein.
I sent my gel slides to different places, none of them could identify them.
problem 1. they said low protein concentration, I could only get silver stain level, no commassie level.
why I can not get enough, TAP tag is two step, final step is calmodulin tag, and elute by 1%SDS, and quite low efficient. and SDS is interfere with MS. and after two step I was left quite little product.
so basically I do not elute the second step, just directly add some 1xSDS loading buffer, boiled, run on gel.
anyone can give me some advice on how to get rid of SDS from SDS loading buffer after I boiled sample??????
or what should I do???????????
I am seeing the protein complex on my gel clearly, but could not know what are they???? drive me crazy!!!!!!!
I try to identify the protein complex by TAP tag. and all seems fine, I see big fat band of my bait protein by silver stain and confirmed by western blot. Then my nightmare started, I spend five months now to try to get MS to identify my protein.
I sent my gel slides to different places, none of them could identify them.
problem 1. they said low protein concentration, I could only get silver stain level, no commassie level.
why I can not get enough, TAP tag is two step, final step is calmodulin tag, and elute by 1%SDS, and quite low efficient. and SDS is interfere with MS. and after two step I was left quite little product.
so basically I do not elute the second step, just directly add some 1xSDS loading buffer, boiled, run on gel.
anyone can give me some advice on how to get rid of SDS from SDS loading buffer after I boiled sample??????
or what should I do???????????
I am seeing the protein complex on my gel clearly, but could not know what are they???? drive me crazy!!!!!!!
from calmodulin where you may need Ca2+ for binding, you can elute with excess of EGTA;
but I would separate the TAP tagged protein complex separate by 2D gel electrophoresis to identify interesting spots...
we recently identified a protein by this method (2D and Ms/Ms); the protein was not to stain by silver (beyond too low amount)some proteins dislike to be stained by silver)
I try to identify the protein complex by TAP tag. and all seems fine, I see big fat band of my bait protein by silver stain and confirmed by western blot. Then my nightmare started, I spend five months now to try to get MS to identify my protein.
I sent my gel slides to different places, none of them could identify them.
problem 1. they said low protein concentration, I could only get silver stain level, no commassie level.
why I can not get enough, TAP tag is two step, final step is calmodulin tag, and elute by 1%SDS, and quite low efficient. and SDS is interfere with MS. and after two step I was left quite little product.
so basically I do not elute the second step, just directly add some 1xSDS loading buffer, boiled, run on gel.
anyone can give me some advice on how to get rid of SDS from SDS loading buffer after I boiled sample??????
or what should I do???????????
I am seeing the protein complex on my gel clearly, but could not know what are they???? drive me crazy!!!!!!!
from calmodulin where you may need Ca2+ for binding, you can elute with excess of EGTA;
but I would separate the TAP tagged protein complex separate by 2D gel electrophoresis to identify interesting spots...
we recently identified a protein by this method (2D and Ms/Ms); the protein was not to stain by silver (beyond too low amount)some proteins dislike to be stained by silver)
The problem is that I could not get enough protein, how is your recovery by EGTA? I found it recovery quite low. right???????
how much material you start with to get enough to stain by 2D????I star with about 100mg Nuclear extract, then finally get about 30ul of sample in 1x SDS buffer, load 20ul on gel. stained with silver stain, can see my big fat sausage protein.
I also used the method, if you start with 13 liter culture of suspention cells, you may get coomassie band of the bait...
I finally go to one step purification.... however, there's also a big problem of background..
I don't think SDS is a big problem, if you run an SDS-PAGE gel.. Silver might be the trouble maker.. If the MS group has MS compatable Silver stain protocol, it would be perfect..
[Oh, my god, 13 litres, quite a lot, enought to fill two incubater!! the maximum I tried is 4 litre. I guess I need to combine couple of times, then send the sample. quite a pain!!!!
how is your one step going then? I tried couple of time with one step, really lots background. not sure is it good enough for MS? and after got hundreds protein, how could I tell which one actrully is due to my protein complex, not because of different runs?
quote name='yeping' date='Jul 11 2007, 02:58 AM' post='104288']
I also used the method, if you start with 13 liter culture of suspention cells, you may get coomassie band of the bait...
I finally go to one step purification.... however, there's also a big problem of background..
I don't think SDS is a big problem, if you run an SDS-PAGE gel.. Silver might be the trouble maker.. If the MS group has MS compatable Silver stain protocol, it would be perfect..
[/quote]
One-step pulls down a lot of background proteins. It's not a good option..
I know from an other group, that they use suspension cells and culture with bio-reactor (something like that..). If the protocol is the same, there should be a protein precipitation step for the final sample. I know they send the precipitation sample directly to MS. ( no PAGE, no staining.) (then the sample will be digested with trypsin in the tube.)
I know from an other group, that they use suspension cells and culture with bio-reactor (something like that..). If the protocol is the same, there should be a protein precipitation step for the final sample. I know they send the precipitation sample directly to MS. ( no PAGE, no staining.) (then the sample will be digested with trypsin in the tube.)
I see. i used TCA precipitation, some collegues said that TCA precipitation lost proteins, I am not sure of that. now I am thinking using Millipore centricon to concentrate my sample, then load all to one lane of gel. then cut to pieces for MS. what do you think
I know from an other group, that they use suspension cells and culture with bio-reactor (something like that..). If the protocol is the same, there should be a protein precipitation step for the final sample. I know they send the precipitation sample directly to MS. ( no PAGE, no staining.) (then the sample will be digested with trypsin in the tube.)
I see. i used TCA precipitation, some collegues said that TCA precipitation lost proteins, I am not sure of that. now I am thinking using Millipore centricon to concentrate my sample, then load all to one lane of gel. then cut to pieces for MS. what do you think
even in mini-gel adapted 2D gel systems, you may load up to 400 mg per strip; for identification there is a rough rule of ~2 pmol protein which means the larger the protein the more mass you need...
I don't know much about TCA. I use chloroform/MeOH to precipitate proteins. And I tested the precipitation efficiency with BSA before performing the real experiment, which was about 50% in my hand..
Millipore centricon may be a good method. (I am not expert....)