SUMO antibody problem (Nikola Wenta)

Suresh Kumar via methods%40net.bio.net (by kumar from lifesensors.com)
Tue May 19 14:52:14 EST 2009



Hi Nikola,

Which SUMO antibody you use to detect SUMOylation and what are your western conditions?
There are three major types of SUMOs (SUMO1, SUMO2 and SUMO3). 
You may need to select the right antibody depending on which isoform of SUMO you used for your in vitro SUMOylation. Antibody concentration and incubation time can also be critical.
There are also Pan speficic antibodies that are supposed to recognize all three isoforms. 

Another method to detect sumoylation is to use in vitro translated (S35 methionine labeled) target protein and detect the slow migrating (sumoylated) target protein band using autoradiography. Since you can already detect SUMOylation using your protein antibody, it's your call if you want to use radio activity. Otherwise all you need is to get a good clean SUMO antibody and work out the conditions.
 
Suresh
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Sent: Monday, May 18, 2009 1:04 PM
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Subject: Methods Digest, Vol 48, Issue 15

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Today's Topics:

   1. RNA labeling at 5' (nada mohamed)
   2. SUMO antibody problem (Nikola Wenta)
   3. RNA labeling at 5' (Tim Fitzwater)


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Message: 1
Date: Sun, 17 May 2009 13:41:42 -0700 (PDT)
From: nada mohamed <nadmsa1 from yahoo.com>
Subject: RNA labeling at 5'
To: methods from magpie.bio.indiana.edu
Message-ID: <288179.46934.qm from web34402.mail.mud.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

Dear all,
 
I am a PhD student and I am studying RNA-Protein interaction. I need a protocol to label my RNA at 5'end with biotin, I found some researches using megascript kit to incorporate biotin and others using Maxiscript I am confused which one is suitable ? and is there any alternative methods, also what is the criteria that must be taken in account for RNA biotin labeling (to be sure that my RNA is not affected by biotin incorporation), I mean the size of RNA the amount of labeled nucleotides. Any help will be appreciated. 




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Message: 2
Date: Mon, 18 May 2009 10:57:41 +0100
From: Nikola Wenta <Nikola.Wenta from nottingham.ac.uk>
Subject: SUMO antibody problem
To: <methods from oat.bio.indiana.edu>
Message-ID:
	<814A9FCB0AD790489679BFBCE6CA5E55303116 from VUIEXCHC.ad.nottingham.ac.uk>
Content-Type: text/plain;	charset="iso-8859-1"

Hi all!
Does anyone have tried to detect SUMOylated protein? I have performed in vitro SUMOylation as well as in vivo. In both cases, I was able to detect my protein and a higher molecular protein band, corresponding to presumably the SUMOylated protein with the protein-specific antibody. However, when I use a SUMO-specific antibody, I fail to detect the SUMOylated protein. Does anyone have any suggestions how to get the SUMO antibody working? Would it be advisable to perform other methods to detect SUMOylation, i.e. MS? 
Thank you for any suggestions!
Niko

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Message: 3
Date: Mon, 18 May 2009 08:48:51 -0600
From: "Tim Fitzwater" <tfitzwater from somalogic.com>
Subject: RNA labeling at 5'
To: <methods from magpie.bio.indiana.edu>
Message-ID: <771BF0D090CE73449F7495D31E0BA0D14E9733 from ELM.sladmin.com>
Content-Type: text/plain;	charset="us-ascii"

 The authors were able to initiate T7 transcription with 4 mM
"5'-biotin-spacer 9-G as the starting nucleotide."  
 
McNamara, J. O., D. Kolonias, et al. (2007). "Multivalent 4-1BB binding
aptamers costimulate CD8 T cells and inhibit tumor growth in mice." J
Clin Invest.

 
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dop
t=Citation&list_uids=18060045
<http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&do
pt=Citation&list_uids=18060045> 

 
Tim


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