General Protocol for N2A Lysis and Western Blotting of Lysates
Combine 1.6 mL of 1x SDS lysis buffer
, 400µL β
-mercaptoethanol, and 20µL of proteaseinhibitor in a 15mL falcon tube2.
After allowing appropriate time for expression in transfected cells (usually 24 hours), Aspiratemedia from each well of a 12-well plate3.
Add 150µL of 1x SDS lysis buffer to each well. Scrape and collect in microcentrifuge tubes.4.
Boil all samples at 100
C for 15 minutes5.
During procedures, keep tubes on ice. Store at -80C.BCA Assay1.
Make up albinum standards, diluted in M-PER/protease inhibitor according to provided protocol2.
Pipette 25µL of standards in technical duplicates into a 96-well clear assay plate3.
Pipette 25µL of samples in technical duplicates into the assay plate.4.
Cover plate and incubate 30 minutes at 32C5.
Uncover plate and load into a 96-well plate reader. Read plate at 260nm6.
Calculate necessary volume for 1µg of total proteinSDS-PAGE1.
Make up MES buffer from 20x MES solution in milliQ H
Load a 4-12% bis-tris gels into a vertical electrophoresis apparatus. Fill with MES buffer.3.
In first lane, load 5µL of benchmark-prestained protein ladder.4.
Load 5µL of each lysate solution into each well in collated duplicate (one for
-tau, one for
Run gel at 200V for 35 minutes or until 10KDa marker is at the bottom of the gelTransfer1.
Make up Tris-Glycine buffer by diluting 5x stock in milliQ H
O and completing to 1X with MeOHso that the final concentration of MeOH is 20%.2.
Cut edges of gel so that all lanes are included3.
Cut two nitrocellulose membranes and incubate in Tris-Glycine buffer until ready to use.4.
Cut whatman gel blot paper slightly smaller than foam pads from the transfer apparatus5.
Prepare “sandwich” as follows:
+, foam pad, Whatman Gel Blot Paper, PVDF membrane, Gel, Whatman Gel Blot Paper,foam pad, -6.
Load into apparatus. In the space provided, load an ice pack. Run gel at 180mA for 75 minutes