IP/Western of HA-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
- 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 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
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