IP- Western of HA-tagged Proteins

Overview
List of reagents is not yet complete

Procedure
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 * develope with ECL kit (Amersham)

Fresh Homogenization Buffer

50mM Tris pH7.4 TM

2.5 ml	1M Tris 1x Complete

5.0 ml	10x Complete TM Cocktail (Protease Inhibitors) 1mM DTT

0.25 ml	200mM (200x)in H20 1mM PMSF

0.25 ml	200mM (200x) in EtOH
 * H2O

42.0 ml

Total

50.0 ml


 * unstable in H2O, therefore use homogenization buffer within 1/2hr after addition 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 ==Notes==

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