Gel storage before immunoblotting
Dr. Hiranya S. Roychowdhury
hroychow at nmsu.edu
Sat Apr 28 10:28:50 EST 2001
Yes, following Cu-staining, you are able to transfer to any matrix of your
choice and the Cu does not transfer to the matrix (naturally!).
Cu-staining is said to be more sensitive that CBB-R staining (~5x). Indeed,
I have been able to detect bands on a Cu-stained gel that are undetectable
in CBB-R stain. I think only Ag-staining is more sensitive. However, since
the staining is negative, the ease of visualization of polypeptide bands
depends on the resolution in the gel.
The staining is *completely* reversible (0.25M EDTA - a little high, IMO
-- in Tris.Cl, pH 9 (100mM)). The polypeptides are not irreversibly *fixed*
and, IMO, there lies the methods usefulness, among others. I usually do
Cu-staining for electroeluting my polypeptide of interest.
The other usefulness of this method is that one can detect the bands very
rapidly once PAGE is done (BTW, seems to work only with SDS-PAGE). So,
during routine purification of protein samples or during standardization of
a protocol, a quick set of minigels (<1h), 5 min Cu-stain followed by
photography allows one to have a permanent record very quickly. The
photographs look as good as those of positively stained gels (like those of
CBB-R or Sypro-orange-stained gels).
At 09:17 AM 4/28/01 -0500, Christopher LaRosa wrote:
>And you can transfer the bands to matrix for immunoblotting?? How
>sensitive is the staining compared to coomassie?
>On Fri, 27 Apr 2001, Dr. Hiranya S. Roychowdhury wrote:
>> I will have to dig up the reference. I do it routinely. Very simple:
>> 1. Rinse the gel, with dist. water, after SDS-PAGE.
>> 2. Soak the gel in 0.3 to 0.4M CuCl2 (made in dist. water). The stain may
>> be reused several times.
>> 3. View/photograph the gel with incandescent lighting, incident at an
>> angle. The exposure timing is to be worked out (usually 1/120th of a sec
>> Yes, the gel can be stored, either in plain (dist.) water or in the CuCl2
>> soln. at 4 C for several weeks, if not months. I have not noticed any loss
>> of resolution or of polypeptide bands after two mnths of storage in CuCl2
>> soln. or in water.
>> Destaining is done with 0.25M EDTA in Tris.Cl, pH 9 (100mM), following
>> which the staining is also lost.
>> At 10:28 AM 4/27/01 -0500, clarosa wrote:
>> >PLease give reference on CU-stain...
>> >"Dr. Hiranya S. Roychowdhury" wrote:
>> >> With Cu-stain, the gel may be stored for several weeks before transfer
>> >> At 10:21 PM 4/11/01 +0100, Choe Woo-seok wrote:
>> >> >After SDS-PAGE, can the gel be stored before electroblotting or is it
>> >> >better to
>> >> >be transfered immediately.
>> >> >In
>> Dr. Hiranya Sankar Roychowdhury
>> College Asst. Prof.
>> Molecular Biology,
>> Dept. of Chemistry & Biochemistry
>> Box 30001 - 3MLS
>> New Mexico State University
>> Las Cruces, NM 88003
>> Lab: (505) 646 4722
>> Office: (505) 646 8256
>> hroychow at nmsu.edu
Dr. Hiranya Sankar Roychowdhury
College Asst. Prof.
Dept. of Chemistry & Biochemistry
Box 30001 - 3MLS
New Mexico State University
Las Cruces, NM 88003
Lab: (505) 646 4722
Office: (505) 646 8256
hroychow at nmsu.edu
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