CAR1 Western Blot:

Samples (add SB )

Electrophoresis: 10% SDS-polyacrylamide gel

Transfer: Immobilon or Nitrocellulose filter 50 volts 3 hr or 30 volts o/n

 Ponceau -S stain filter 

1xTBS-T wash

Block membrane in 3%BSA/TBS-T for 1 hour at RT

Wash membrane  in 1xTBS-T

Incubate membrane in 1/1000 diluted Anti-CAR1 for 1hour at RT

Wash membrane 3x10 minutes in 1xTBS-T

Incubate membrane in 1/10000 diluted Anti-rabbit/TBS-T for 1hour at RT

Wash membrane 3x10 minutes in 1xTBS-T

Mix equal amounts of Enhanced Luminol Reagent and Oxidizing Reagent

Incubate membrane in detection mixture for 1 minute at RT

place membrane in plastic sheet

Expose membrane to film for 10" to 1’

Develop film

Pre-absorb CAR1 Antibody

5 x e8 cells (1 mg protein) : DB wash

Resuspend in 5ml DB

Fix : 45ml 50% methanol/DB ( ice cold ), Rock for 20 minutes at 4c

Wash cells in DB 3 times

Block cells in 3%BSA for 30 minutes at RT

Spin down

Incubate cells in 50ml 1:1000Anti-CAR1/1xTBS-T, rock in cold room  overnight

Spin down

Save supernatant at 4c

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This page was last edited 06/26/2003