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Western Blotting with Alkaline Phosphatase Conjugates SAMPLE PREPARATION For Protein Concentration Determination of Cell Culture 1.Decant

REAGENTS ECL Western blotting kit (Amersham Life Science; cat# RPN2108): contains second antibodies for both mouse and rabbit, substrate an

Materials Blot Cell BA 83 0.2 µm pore nitrocellulose sheets Buffer, PBS-Tween 20 Antigenic proteins, antibodies, and horseradish peroxid

he following tips can be used to overcome the most common problems encountered during Western blotting. Smeared Pattern or Distorted Bands

Wash XCell box, blotting module and trays with soap and water before proceeding. Reagents: Transfer buffer 25 mM Tris HCl, 190 mM Glycin

Semi-dry Transfer Apparatus Biorad, Cat# 170-3940, or equivalent Power Supply 0 - 100 VDC (adj. current to 1 Amp) Immobilon-P transfer mem

1. Treat cells. For PC12 or NIH 3T3 cells a moderately confluent 60 mm plate is used. 2. Aspirate media. Rinse cells with PBS, aspirate. 3

REAGENTS ECL Western blotting kit (Amersham Life Science; cat# RPN2108): contains second antibodies for both mouse and rabbit, substrate an

Blotting with BSA 1. Blot in 5% mile/ 1X PBST (or 1X TBST) --60'/Rt 2. Wash 2X for 5' in 1XPBST 3. 10Ab in 1% BSA/1X PBST --60'/Rt 4. Wa

Running Protein Gels Solutions 10X Running Buffer (0.25 M Tris, 1.92 M glycine, 1% SDS) 121 g Tris 577 g glycine 40 g SDS ddh20 to 4 L