[proteomics] Re: Yan: Hello from Proteomics List Manager
- From: Mavi Gozler <mavigozler@xxxxxxxxx>
- To: proteomics@xxxxxxxxxxxxx
- Date: Tue, 16 Dec 2008 10:24:53 -0800 (PST)
Hello Emin,
There could be several problems with your identification of your molecular
weight markers. I don't know about SilverQuest, but I am assuming its an
acid-based method of silver staining. The amine-based methods are said not to
be MS-compatible.
Are you making sure that you are de-staining (with the standard mix of
ferricyanide and thiosulfate) your bands/spots before doing your in-gel
digestion?
What about reductive alkylation subsequently in situ (with DTT/iodoacetamide)
before trypsin/protease digestion?
How does your MALDI spectrum look? If you are using fingerprinting against a
database search as the mode of identification, it really does help if you have
a mass error of 50 ppm or less and that your MALDI is very well calibrated
within required tolerances, or identifications are made with very little
certainty. If your spectrum is quite noisy, you will probably benefit from
using ZipTips. If you are getting sequence tags with induced fragmentation
(PSD?), this will raise your confidence level in doing identification.
As far as the "empty" areas of the gel, what are you seeing as a result? A
noisy spectrum is not uncommon. Unfortunately the query software we are forced
to use is not so well written that it refuses to give a list of possible
protein IDs and say "no proteins identified." Instead it will give a list of
proteins usually with a very low confidence level, but no hits should be
acceptable when confidence levels are below even 50%.
Rabilloud and co-workers have done considerable work in silver staining
chemistry. We like the method used by Rabilloud, Carpentier, Tarroux (1988)
Electrophoresis 9:
288-290.
Mitch
________________________________
From: Emin Oztas <emino55@xxxxxxxxx>
To: proteomics@xxxxxxxxxxxxx
Sent: Monday, December 15, 2008 11:42:11 AM
Subject: [proteomics] Yan: Hello from Proteomics List Manager
Hello again to all,
I have a problem with silver stainining. I did 2D IEF with a Immuno precipated
protein from a cell line. Silver staining kit (SilverQuest, invitrogen) says
that their stain is mass spec compatible. But we tested it and marker bands
were not recognized by MALDI. We also found false or wrong results from empthy
area of the gel piece.
If anybody have any experience with SilverQuest silver staining kit please send
recommandations.
Dr. Emin Oztas
cand
--- 14/12/08 Pzr tarihinde Mavi Gozler <mavigozler@xxxxxxxxx> şöyle yazıyor:
> Kimden: Mavi Gozler <mavigozler@xxxxxxxxx>
> Konu: [proteomics] Hello from Proteomics List Manager
> Kime: proteomics@xxxxxxxxxxxxx
> Tarihi: 14 Aralık 2008 Pazar, 12:49
> I am hoping to see more people subscribe to this list and
> start posting messages to it.
>
> Any message related to proteomics is welcome:
>
>
> * Questions seeking answers to problems in proteomics
>
> * Answers to questions providing solutions to problems
> in proteomics
>
> * Your own "newsletter" with maybe things you
> have read and which you recommend the list to read
>
>
> * And yes, this list allows product vendors/makers to
> advertise their
> proteomics-related products, so long as they put
> "PRODUCT AD" or
> "ADVERTISEMENT" as the leading phrase in the
> Subject header (so that
> list members can filter it out, if they don't want to
> read it)Remember,
> proteomics is a broad discipline with overlap in genomics,
> part of "functional genomics." Much of anything
> in molecular and cellular biology and biochemistry would be
> of interest to the list.
>
> So let's please make this list lively.
>
> Tell your friends to subscribe by creating a new email
> message and filling in the following headers:
>
> To: proteomics-request@xxxxxxxxxxxxx
> Subject: subscribe
>
> They can also put the word "subscribe" in the
> message body as well as Subject header.
>
> Best wishes for your continuing success in all your
> efforts,
>
> Mitch Halloran
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