IP-Western of A-tagged Proteins

This protocol was developed by Michael Y. Degtyarev, Ph.D. in the Conklin Lab at The Gladstone Institute of Cardiovascular Disease, April 1998.

1. Homogenization.

• Place tissue (50-500 mg) in cold 1 ml homogenization buffer in 15 ml round bottom Falcon tube (cat# 2059). Homogenize with a polytron homogenizer. Transfer the homogenate (1-1.5ml) into a 1.5 ml eppendorf tube. Measure a protein concentration.

2. Solubilization.

• 750 microliters of 2xIP buffer + X microliters of H2O + Y microliters of homogenate (1 mg of total protein) => Total V=1.5 ml
• sonicate for a few seconds if the sample is very viscous
• incubate 30 min. at 4 degrees C on a rotator
• spin at max speed table centr. for 5 min.
• transfer a supernatant to a new 1.5 ml tube

3. Immunoprecipitation.

• to supernatant add 10 microliters (100 ng/ ml) of polyclonal rabbit anti-alpha q/11 antibody (C-19, Santa Cruz Biotech., cat # SC-392) and 15 microliters of Protein A-agarose 50% suspension (Sigma)
• incubate overnight at 4 degrees C on rotator
• spin down beads for 1 min at 4 degrees C 3,000 rpm
• discard supernatant using a vacuum (be careful not to remove beads)
• wash beads 2x750 microliters with wash buffer: add wash buffer to beads, invert a few times and spin down beads for 1 min at room temperature at 3,000 rpm, discard supernatant
• add 50 microliters of 1x electrophoresis sample buffer (Novex) to the beads pellet
• vortex to resuspend beads; boil sample for 5 minutes
• vortex beads; spin down beads for 2 min at room temperature 5,000 rpm
• transfer supernatant to new tube using flat tips or load supernatant on gel using flat tips.
• Pipette carefully and try not to disturb bead pellet.

4. Western blotting

• run a 10-12% gel; transfer to nitrocellulose (NC)
• incubate NC in 5% dry milk in PBS for 30-60 min.; rinse in PBS for 1 min.
• incubate NC in anti HA-HRP antibody (Boehringer Mannheim) for 1-3 hours using 5,000-10,000 dilution in PBS-T with 1%BSA
• wash 2x15 min. in PBS-T
• develop with ECL kit (Amersham)

• unstable in H2O, therefore use homogenization buffer within 1/2hr afteraddition of PMSF

2x IP buffer:

• 100 mM Tris-HCl pH 7.4

• 300 mM NaCl

• 2% NP-40

• 1% NadeoxyCholate

• 0.2% SDS

Wash Buffer:

• 1x TBS (50mM Tris-HCl, pH 7.4, 150mM NaCl) + 1/20 of 2xIP buffer

Sample Buffer:

• 2x sample buffer, 5% beta-mercaptoethanol

• (Tris-Glycine SDS) dilute to 1x with H20

10x Complete Cocktail (Boehringer):

Dissolve 1 tablet (Cat# 1697498) in 5 ml H2O or 1 mini-tablet (Cat# 1836153)in 1 ml H2O