SPI-Mark™ Colloidal Gold Reagents
Immunostaining of Protein Gel Transfers (Western blots)
(A) Perform a Coomassie blue stain on a strip of the gel to determine the whole
protein staining pattern. Alternatively use PROTOGOLD (from BBI) on a
separate blotted membrane to give a total protein stain.
(B) Transfer the protein pattern to the immobilising matrix by established
blotting procedures.
(C) Wash the membrane thoroughly in PBS.
(D) Block the unoccupied sites by immersing the membrane in 10% BSA for 30 min.
(E) Transfer the membrane to the primary antibody diluted in buffer A. Incubate
for 1 hour while agitating.
(F) Wash the membrane very thoroughly in buffer A while agitating for 30 min.
(G) Immerse the membrane in gold labelled second antibody diluted in buffer A
while agitating. Dilution of the antibody should be determined as in 4.3.
Incubate for 1 hour.
(H) Wash the membrane very thoroughly in buffer A for 30 min and then in
distilled water for 30 min while agitating.
Gold conjugates of 20nm will appear visible directly as red stains. For 1nm
gold conjugates silver enhancing is necessary.
(I) Immerse the membrane in fresh silver enhancing solution and agitate for 10-
20 min. Wash thoroughly in water to stop the reaction. The protein bands will
appear as black lines.
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Thursday May 17, 2012
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