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santacruz  G蛋白PLUS-瓊脂糖Protein G PLUS-Agarose

SANTA CRUZ BIOTECHNOLOGY, INC.
Protein G PLUS-Agarose
Immunoprecipitation Reagent: sc-2002
Santa Cruz Biotechnology, Inc. 1.800.457.3801 831.457.3800 831.457.3801 Europe +00800 4573 8000 49 6221 4503 0 www.scbt.com
PRODUCT
Protein G PLUS is provided as an agarose conjugate for use in immunoprecipitation
only. The product is provided as 0.5 ml agarose in 2.0 ml PBS buffer
with 0.02% azide. Protein G PLUS-Agarose is pre-blocked with BSA to reduce
non-specific immunoglobulin binding. Sufficient product is provided for 100
immunoprecipitation reactions, to be used at 20 μl resuspended volume per
reaction.
SPECIFICITY
Protein G PLUS-Agarose is suitable for immunoprecipitation of mouse IgG1,
IgG2a, IgG2b and IgG3, rat IgG1, IgG2a, IgG2b and IgG2c, rabbit and goat polyclonal
Abs, and human IgG1, IgG2, IgG3 and IgG4.
PROCEDURE
? Incubate cultured cells （80-90% confluent monolayer in 100 mm cell culture
plate, or approximay 2-5 x 107 suspension cells in flask） in methioninefree
medium containing 5% dialyzed fetal calf serum for 1 hour at 37° C.
The same procedure can be used for cells labeled with other radioactive
amino acids （e.g., 14C or 3H） or with γ32P-orthophosphate. Cell labeling must
be carried out in medium lacking the relevant amino acid or in phosphatefree
medium.
? Remove medium and replace with 3 ml methionine-free medium containing
5% dialyzed fetal calf serum and 100 μCi/ml 35S-methionine. Incubate 1
hour at 37° C. For some proteins a longer labeling period （up to 18 hours）
is preferable.
? Carefully remove radioactive medium with Pasteur pipette and wash cell
monolayer with PBS.
? Add 3 ml ice cold RIPA buffer to cell monolayer and incubate at 4° C for
10 minutes. For suspension cells, add the RIPA buffer to washed cell pellet
in a 15 ml conical centrifuge tube.
? Disrupt cells by repeated aspiration through a 21 gauge needle and transfer
to a 15 ml conical centrifuge tube.
? Wash cell culture plate with additional 1.0 ml ice cold RIPA buffer and
combine with original extract.
? Pellet cellular debris by centrifugation at 10,000 xg for 10 minutes at 4° C.
Transfer supernatant to a fresh 15 ml conical centrifuge tube on ice.
Preclear lysate （optional step） by adding 1.0 μg of the appropriate control
IgG （normal mouse, rat, rabbit or goat IgG, corresponding to the host
species of the primary antibody）, together with 20 μl of resuspended
volume of Protein G PLUS-Agarose. Incubate at 4° C for 30 minutes.
? Pellet beads by centrifugation at 2,500 rpm （approximay 1,000 xg） for
5 minutes at 4° C. Transfer supernatant （cell lysate） to a fresh 15 ml conical
centrifuge tube on ice.
? Transfer 1 ml of the above cell lysate, or approximay 100-500 μg total
cellular protein, to a 1.5 ml microcentrifuge tube. Add 1-10 μl （i.e., 0.2-2 μg）
primary antibody （optimal antibody concentration should be determined by
titration） and incubate for 1 hour at 4° C.
? Add 20 μl of resuspended volume of Protein G PLUS-Agarose. Cap tubes
and incubate at 4° C on a rocker platform or rotating device for 1 hour to
overnight.
? Collect immunoprecipitates by centrifugation at 2,500 rpm （approximay
1,000 xg） for 5 minutes at 4° C. Carefully aspirate and discard radioactive
supernatant.
? Wash pellet 4 times with 1.0 ml RIPA buffer （more stringent） or PBS （less
stringent）, each time repeating centrifugation step above.
? After final wash, aspirate and discard supernatant and resuspend pellet in
40 μl of 1x electrophoresis sample buffer.
? Boil samples for 2–3 minutes and analyze 20 μl aliquots by SDS-PAGE and
autoradiography. Unused samples may be stored at -20° C.
? Optional: After boiling, samples may be centrifuged to pellet the agarose
beads followed by SDS-PAGE analysis of the supernatant.
SELECT PRODUCT CITATIONS
1. Medema, R., et al. 1998. p21waf1 can block cells at two points in the cell
cycle, but does not interfere with processive DNA-replication or stressactivated
kinases. Oncogene 16: 431-441.
2. Marzo, I., et al. 1998. Bax and adenine nucleotide translocator cooperate
in the mitochondrial control of apoptosis. Science 281: 2027-2031.
3. Song, Y., et al. 2011. Ligand-dependent corepressor acts as a novel corepressor
of thyroid hormone receptor and represses hepatic lipogenesis in
mice. J. Hepatol. 56: 248-254.
4. Beard, R.S., et al. 2012. Metabotropic glutamate receptor 5 mediates
phosphorylation of vascular endothelial cadherin and nuclear localization
of β-catenin in response to homocysteine. Vascul. Pharmacol. 56: 159-167.
STORAGE
Store at 4° C, do not freeze; stable for one year from the date of shipment.
RESEARCH USE
For research use only, not for use in diagnostic procedures.
Immunoprecipitation agarose conjugates are pre-blocked with BSA to reduce non-specific immunoglobulin
binding and are provided at a concentration （0.5 ml agarose/2.0 ml） suitable for use at 20 μl per
immunoprecipitation reaction. Number of reactions: 100.

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