Western Blotting With AMRESCO BIO-BLOCK - Code E671, E667, E576, E578

Directions for use 

 

1-  The protein containing sample solution should be buffered using:

  • SDS to denature the protein and create a consistent charge to mass ratio
  • Glycerol to allow the sample to settle in the electrophoresis gel
  • Reducing agent if necessary, to reduce disulfide bonds (DTT or (-Mercaptoethanol)
  • Tracking dye
2- Separate the proteins by MW in an SDS-Polyacrylamide gel.
3- Transfer the proteins to a nitrocellulose, PVDF, or nylon membrane.
4- Using AMRESCO BIO-BLOCKª, soak the membrane for 40-60 minutes at room temperature (while shaking), to block non-specific sites. The addition of Tween-20 is recommended in a final concentration of 0.05%.
5- Dilute the primary antibody (optimal antibody dilutions must be developed for each specific antibody) in AMRESCO BIO-BLOCKª (with 0.05% Tween-20) and add it to the membrane.  Incubate the membrane in this solution for 1 hour with shaking.
6- Using AMRESCO Phosphate Buffered Saline, wash the membrane with 5-6 five minute washing cycles. Tween-20 may be added (0.05% final concentration) to reduce background, but this may also cause protein dissociation from the membrane.
7- Dilute the conjugated secondary antibody in AMRESCO BIO-BLOCKª (0.05%      Tween-20) and add to the membrane, incubating for 1 hour at room temperature with shaking.
8- Repeat membrane washes as described in Step 6.
9- Develop the blot using the appropriate detection methods.

 

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