|
Step 1:
|
Combine affinity matrix and
IP Buffer in a microfuge tube at a 1:5 dilution. Add tagged protein
to mixture. Gently rock aliquots for one hour at 4C.
|
|
Step 2:
|
Centrifuge mixture at 10,000g
for 20 seconds at 4C. Remove supernatant without disturbing
beads. Add an equal volume of IP Wash Buffer to the beads and
re-suspend matrix. Gently rock aliquots for 20 min. at 4C.
Keep a portion of the supernatant from each rinse step to use
in Western blot analysis.
|
|
Step 3:
|
Repeat step 2 four times.
|
|
Step 4:
|
Elute the bound protein with
the appropriate epitope peptide at 1 mg/ml concentration in 50
mm Tris HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA (pH 8.0). Resuspend
beads, incubate, centrifuge and withdraw supernatant as in step
2, repeating for a total of four elutions. Recover as much of
the eluate as possible at each stage.
|
|
Step 5:
|
For each elution sample, prepare
at 1:1 dilution with reducing gel loading buffer. Boil tubes
for 3 min.
|
|
Step 6:
|
Analyze the supernatant samples
by Western blot.
|
|
|
IP Buffer
50 mm Tris HCl (pH 7.5) 150 mM NaCl 0.1% Tween
20 (or 0.1% Nonidet P 40) 0.5% BSA 1 mM EDTA (pH 8.0) 1mM b-mercaptoethanol
(see Considerations) 1mM phenlymethylsulfonyl fluoride (PMSF)
or protease inhibitors of choice
|
Wash Buffer
50 mm Tris HCl (pH 7.5) 150 mM NaCl 0.1% Tween
20 (or 0.1% Nonidet P 40) 1 mM EDTA (pH 8.0) 1mM phenlymethylsulfonyl
fluoride (PMSF) or protease inhibitors of choice
|
|
|
Considerations
:
-
The ingredient b-mercaptoethanol is not
always necessary but is included in the immunopurification buffer
to improve the solubility of the proteins in the lysate. At the
concentration indicated, it should not reduce antibodies. DTT
may be used as a substitute.
-
The suggested immunopurification buffer
is by no means the only one that will work. A wide variety of
other common physiologic buffers may be employed instead.
-
At step 4, if elution is not desired,
bound protein may be visualized on a gel by resuspending beads
directly in reducing gel loading buffer, boiling for 5 minutes,
and loading the supernatants on a gel for analysis. Note that
the loading buffer may release a portion of the antibodies from
the affinity matrix.
|
