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