![]() ![]() Small needle-point markings can be added to membrane in-line with color markers which reduce in intensity following subsequent incubation and washing steps.This precast gel contains 18 lanes with a loading capacity of 10-40 ug protein in up to 30 ul per well. ![]() Procedural notes for this Western Blot Method: Mix 1 ml each of ECL reagents in a foil-wrapped tube and add to membrane for 5 min prior to imaging on ChemiDoc MP imager (Bio-Rad).Wash membrane three times in TBS with 0.2% Tween20 at 4☌ with rocking, for 10 min each time.Incubate with diluted secondary antibody 1:2500 in 5 ml TBS with 0.2% Tween20 at 4☌ with rocking for 1 h.Pour off blocking buffer and add diluted anti-RCAN1 antibody 1:200 in 5 ml TBS with 2.5% skim milk power and 0.05% Tween20.Incubate transfer membrane in blocking buffer for 1 h at 4☌ with rocking.Remove transfer membrane from cassette, taking care to snip one corner to ensure orientation.*.Close cassette and run in machine (standard minigel program for 30 min).Use a glass rod to roll across the “sandwich” to remove any air bubbles. Assemble transfer “sandwich” by placing down soaked blotting paper, transfer membrane, gel and blotting paper onto open transfer cassette (Turbo Blot transfer unit Bio-Rad).Remove gel from tank and place in transfer buffer.Activate transfer membrane (also cut to size) by dipping in methanol, then soak in transfer buffer for approx. While gel is running, soak two pieces of blotting paper (cut to the same size as the gel) in transfer buffer (approx.Load gel with samples and include ladder in one lane.Heat samples to 95☌ for 2 min and spin briefly to ensure contents are at the bottom of the tube.In an Eppendorf tube add protein sample (30 µg) to 10 µl 4X SDS-PAGE loading buffer and add water to a final volume of 40 µl.Assemble gel in tank and fill with running buffer.* Unwrap precast gel and rinse wells three times with running buffer.Blotting paper (ProteanXL, #1703966 Bio-Rad).Immun-Star WesternC Chemiluminescence reaction solutions (#170-5070 Bio-Rad). ![]()
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