2012 Promega catalogue

Life
Science
Catalog
2012
Worldwide Contact List
Section
Contents
Table of
Contents
Cell Signaling
Cell Signaling Antibodies
Product Size Cat.# Price ($)
Anti-pS 473 Akt pAb 40 µl G7441 500.00
Immunocytochemical staining of Akt in embryonic rat brain cells.
Embryonic (day 17) rat brain cells were collected and treated with
20ng/ml each of EGF and FGF. Anti-pS 473 Akt pAb was used at a 1:50
dilution. Positive cells were visualized using a donkey anti-rabbit, Cy ® 3conjugated
secondary antibody. Nuclei were stained using DAPI. Protocols
developed and performed at Promega.
66
A. 1 2
B.
1 2
2769TA10_9A
– Phospho-Akt – Akt
Detection of phosphorylated Akt by Western blot analysis with AntipS
473 Akt pAb. Panel A. NIH/3T3 total cell extract (10μg per lane) was
resolved by polyacrylamide gel electrophoresis and blotted onto nitrocellulose.
Lane 1, untreated cells; lane 2, cells pretreated with PDGF (Invitrogen) at
50ng/ml for 20 minutes. Anti-pS 473 Akt pAb (Cat.# G7441) was used at a
1:2,500 dilution. The blot was probed with Donkey Anti-Rabbit IgG (H + L),
HRP, Anti-ACTIVE ® Qualified pAb (Cat.# V7951) at 1:10,000 dilution followed
by chemiluminescent detection. Panel B. A pan-Akt pAb (New England
Biolabs) reveals total Akt in both stimulated and unstimulated NIH/3T3 cell
extracts. Secondary antibody and detection methods were the same as those
used in Panel A.
2810TA10_9A
Product Size Cat.# Price ($)
Anti-ACTIVE ® CaM KII pAb, Rabbit, (pT286 ) 40 µl V1111 671.00
kDa
250 –
98 –
64 –
50 –
36 –
30 –
16 –
6 –
4 –
0.5µg 2.5µg 5µg 12.5µg 25µg
+ – + – + – + – + –
1 2 3 4 5 6 7 8 9 10 11
– Phospho
T 286
CaM KII
Detection of CaM KII by Anti-ACTIVE ® CaM KII pAb in Western
analysis of rat brain homogenate. Lanes 2, 4, 6, 8 and 10 contain
autophosphorylated (+) brain cytosolic protein in the amounts shown;
lanes 3, 5, 7, 9 and 11 contain nonphosphorylated (–) brain cytosolic
protein in the amounts shown. The presence of the autophosphorylated
CaM KII was detected using the Anti-ACTIVE ® CaM KII pAb diluted 1:5,000.
Panel A Panel B
Panel C
Immunocytochemical detection of autophosphorylated CaM KII in
PC12 cells with Anti-ACTIVE ® CaM KII pAb. PC12 cells were adhered
to slides coated with collagen, fixed in 10% paraformaldehyde for 30
minutes, rinsed in PBS and permeabilized in methanol for 10 minutes
at –20˚C. The cells were then blocked in 1% BSA in PBS for 45 minutes
followed by 2% horse serum in PBS for 60 minutes. Cells were incubated
overnight at 4˚C with (Panel A) Ab alone, (Panel B) Ab preincubated with
a nonphosphorylated CaM KII peptide fragment (1μg/ml) or (Panel C) Ab
preincubated with a phosphorylated CaM KII peptide fragment (1μg/ml). The
Anti-ACTIVE ® CaM KII pAb was used at 1:500 dilution and preincubated
with peptide for 8 hours at 4˚C. After incubation with the Anti-ACTIVE ® CaM
KII pAb or Ab/peptide mixture, the cells were rinsed in PBS and incubated
with a donkey anti-rabbit FITC-conjugated secondary Ab (1:500) for 60
minutes at room temperature.The results demonstrate that preincubation
of the Anti-ACTIVE ® CaM KII pAb with phosphorylated CaM KII peptide
completely abolishes immunostaining (Panel C), but preincubation with
nonphosphorylated CaM KII peptide has no effect on immunostaining (Panel A
versus Panel B). Protocols developed and performed at Promega.
For complete and up-to-date product information visit: www.promega.com/catalog
2055TA02_8A
2057TA02_8A