Western Blot
From Genome@Yale
Transfer Buffer 1L 48mM Tris Base 5.82g 39mM Glycine 2.93g 20% methanol 200mL 0.0375% SDS 3.75mL 10%SDS
PBST 1L PBS 100mL 10X dH2O 900mL 0.05% Tween-20 500uL 100%
Blocking Buffer 50mL PBST 50mL 5% dried milk 2.5g
- Transfer to nitrocellulose or PDVF membrane by semi-dry transfer apparatus, 15V for 45 minutes at room temperature
- Incubate filter in Blocking Buffer 1 hour at room temperature, rocking
- Dilute primary antibody in Blocking Buffer and incubate the filter in the solution 1 hour to O/N at 4°C shaker
- Wash 4X with 10mL PBST, 10 minutes each at RT on rocker
- Incubate 1 hour at RT rocker in 10mL of diluted secondary HRP-conjugated antibody
- Wash 4X with 10mL PBST, 10 minutes each at RT on rocker
- Develop the blot with ECL or TBS
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