1.0 1237084852495 OMERO.editor PROTOCOL_ARCHIVE Standard western blot procedure Protocol adapted from http://openwetware.org/wiki/Western_Blot_(immunoblot). Prior to running the western blotting experiment, consult with the vendor in order to determine if they have optimized the titration for the antibody you just purchased. Most vendors should offer some guideline for how to use the antibody. Value your time and effort first and foremost. If the experiment comes out strange, make the call or email the vendor for assistance. Your time is the most valuable element to the procedure! 1236819629094 Will Moore Load Gel Load [[0]] prepared from fresh buffers. Resolve proteins on the gel voltage gradient. param 0 OPTIONAL ENUMERATION 50 ug tissue extract 50 ug whole cell lysate 50 ug tissue extract 20 ug of nuclear extract SINGLE_STEP Run lane number [[1]] with sample [[2]]. Lane number 1 OPTIONAL TEXT 1 2 3 4 5 6 7 8 Sample name 2 OPTIONAL TEXT control siRNAi INCENP siRNAi Aurora-B siRNAi Survivin siRNAi control siRNAi INCENP siRNAi Aurora-B siRNAi Survivin siRNAi Duplicate lanes Ran each sample twice. Once on each half of the blot, so that the gel could be cut in half and blotted with 2 different antibodies. STEP_GROUP Transfer proteins to a nitrocellulose or PVDF membrane and block in fresh 5% milk TTBS 2 hr, r.t. or overnight at 4C. Perform 3 shake rinses and drain until no residual milk appears to stream off the wash buffer. SINGLE_STEP Antibody incubation If using different antibodies for different lanes [[3]] cut membrane accordingly. Incubate the primary antibody [[4]] 1:200-20000 in fresh 5% milk TTBS for 120 minutes room temperature. Perform 3 shake rinses followed by wash with cold TTBS 4X for 5 minutes each wash. Incubate the secondary antibody [[5]] in fresh 5% milk TTBS for 60 minutes at room temperature. Perform 3 shake rinses followed by wash with cold TTBS 4X for 5 minutes each wash. lanes 3 OPTIONAL TEXT 1-4 5-8 primary antibody 4 OPTIONAL TEXT Anti-INCENP Anti-H3 param 5 OPTIONAL ENUMERATION 1:2000 anti-mouse 1:5000 anti-rabbit 1:2000 anti-mouse 1:2000 anti-mouse 1:5000 anti-rabbit 1:5000 anti-goat STEP_GROUP Perform ECL using a suitable chemiluminescent reagent SINGLE_STEP Optimize protein signal with multiple exposure times. SINGLE_STEP