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EU2: Affinity purified rabbit
anti-GFP polyclonal antibody |
Suggested Unit
Size: |
25 ug (0.025 mg) |
Suggested Unit Price: |
$295 US |
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Approximately 5.0 mg IgG / ml serum (assayed
by binding to protein A agarose). Concentration may vary between
bleeds.
Product
Use |
Yes
/ No |
Recommended
Use |
Western
Blot (WB) |
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dilute
1/1000 to 1/2500 in 1X BLOTTO or 3% BSA in PBS
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Immunoprecipitation
(IP) |
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1.0 ul crude serum per 10
cm tissue culture dish. Use 10 uL protein A agarose CL4B
(Amersham-Pharmacia) to precipitate immune complex |
IP-Western protocols |
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1.0 uL crude serum per 10
cm tissue culture dish. Use 10 uL protein A agarose CL4B
to precipitate immune complex. Run samples on 12.5% SDS-PAGE,
transfer proteins to PVDF membrane, probe membrane with
GFP antibody at 1/1000 to 1/2500 dilution, use protein
A-HRP conjugate as secondary reagent in ECL or ECL Plus
detection kit (Amersham-Pharmacia) as recommended |
Immunohistochemistry &
Immunofluorescence |
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Dilute anti-GFP 1/200 to 1/1000
in 4% normal donkey serum (Jackson Immunologics). Incubate
for 1 hour at 37oC in humidified atmosphere.
Process with secondary antibody as recommended by supplier.
See immunofluorescence photograph (above)
for typical results. |
For additional information please consult the following
references:
- McCabe, James B. and Berthiaume, Luc G. (1999) “Functional
roles of NH2-terminal domains in subcellular localization”
Mol. Biol. Cell 10, 3771-3786).
- Renata Jasinska, Qiu-Xia Zhang, Carlos Pilquil,
Jay Dewald, Deirdre A., Dillon, Luc G. Berthiaume,
George M. Carman and David W. Waggoner, David N. Brindley
“Lipid phosphate phosphohydrolase-1 degrades
exogenous glycerolipid and sphingolipid phosphate
esters”(1999) Biochem. J. 340, 677-686.
- Zhang, Qiu-Xia, Pilquil, Carlos, Dewald, Jay, Berthiaume,
Luc G., and Brindley, David N. Identification of structurally
important domains of lipid phosphate phosphatase-1:
implications for sites of action” (2000) Biochem.
J. 345, 181-184.
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Crude antibody (serum) can be stored for at least 24 months
at -80oC without apparent loss of activity. This
antibody can be frozen and thawed a few times but avoid multiple
unnecessary freeze-thaw cycles. Once thawed, the antibody
is stable up to two weeks at 4oC.
This polyclonal anti-GFP antibody allows detection of 2 to
5 ng recombinant GFP in ECL or ECL Plus (Amersham-Pharmacia)
detection protocols in under one minute of exposition to film
(when used with antibody that is kept at 4oC. Each bleed is
tested for activity in a Western blotting procedure.
N.B. Typically, exposure times required to
detect 2 ng - 5 ng GFP are achieved in as little as one to
five seconds when the anti-GFP antibody is prepared fresh
from the –20 or –80 oC stocks and used at a 1/2500
dilution using the procedure described in Klapper et al.
(1992) Biotechniques, 12, 651-654.
We have over 150 ml of frozen anti-GFP antiserum in the –80oC
freezer. –80oC shelf-life so far is over three years
without apparent loss of signal in our Western blot assays.
Please note that stocks/supply can be increased upon request.
Please note that we carry out only a small inventory (about
2-5 mgs) of affinity purified rabbit anti-GFP polyclonal antibody
but larger amounts can be prepared up on demand.
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