Methods

Immunoblotting Method

Samples

Protein extraction performed in a buffer consisting of: Tris 10mM, EDTA1mM, proteases inhibitors pH 7.4 (e.g. Protease inhibitor Kit of Quartett, Germany or cOmplete of Roche-Sigma (cat. Nr. 04693159001 Roche).

The extraction of the proteins from the tissue is done in the above buffer by sonication (2 x 10 seconds on ice, followed by centrifugation at 14’000 rpm, during 10 min at 4°C).

Sample loading

30 to 50 ug proteins / lane

Controls: cerebellum or muscle (EDL: extensor digitorum longus)

SDS-gel: 12 or 15% SDS-polyacrylamide gel

Transfer of the protein

Semi-dry for 1h, with 60mA / membrane.

Transfer buffer: Tris Base 25mM,Glycine 192mM (pH8.3) 200 ml methanol for 1L.

Primary and secondary antibodies: primary antibody at a dilution of 1:2’000 to 1:10’000 in 2% milk/TBST, 4°C overnight.

Secondary antibodies (e.g. rabbit) Sigma A0545, diluted 1:10’000, 2h at room temperature, 2& milk/TBST

Detection system

Luminata forte (Millipore) or WesternBright Quantum of Advansta

Important for taking pictures with an automatic camera: if you have very strong bands in the upper molecular weight, hide them with a sheet of black paper and repeat the detection there were at first sight you had no band (e.g. 12 KDa).

Swant sells the best antibodies against calcium binding proteins.

New prices from August 1, 2017

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