2012 Promega catalogue

Life
Science
Catalog
2012
Worldwide Contact List
Section
Contents
Table of
Contents
Cell Signaling
A. 1 2
B.
166
1 2
– Phospho-Akt – Akt
Detection of phosphorylated Akt by Western blot analysis with
Anti-pS 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.
Anti-ACTIVE ® CaM KII pAb, Rabbit, (pT 286 )
Product Size Cat.# Price ($)
Anti-ACTIVE ® CaM KII pAb, Rabbit, (pT286 )
For Research Use Only. Not for Use in Diagnostic Procedures.
40 µl V1111 671.00
Description: This polyclonal antibody (pAb) is specific for the multifunctional
calcium/calmodulin-dependent protein kinase CaM kinase II (CaM KII) that is
phosphorylated on threonine 286 (pT 286 ). The Anti-ACTIVE ® CaM KII pAb was
raised against the phosphothreonine-containing peptide.
Features:
• Specificity: Preferentially detects CaM KII autophosphorylated on
threonine 286.
• Immunogen: Threonine-phosphorylated peptide corresponding to the
phosphorylated Thr 286 region of the mammalian calcium/calmodulindependent
protein kinase.
• Antibody Form: Affinity-purified rabbit IgG; supplied in 10mM sodium
phosphate (pH 7.4), 20mM NaCl.
• Value: When used at the recommended dilution of 1:5,000, it generates
up to 200ml of blotting solution sufficient for 20 Western blots of 10ml
each.
Storage Conditions: Store at –20°C.
Protocol Part#
TB264
Anti-ACTIVE ® CaM KII pAb, (pT 286 ) and Anti-ACTIVE ® Qualified
Secondary Antibody Conjugates Technical Bulletin
2810TA10_9A
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. Staining was visualized with a Zeiss ® fluorescence
microscope. 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