2013 Promega catalogue
2013 Promega catalogue
2013 Promega catalogue
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Cell Signaling<br />
Anti-ACTIVE ® CaM KII pAb, Rabbit, (pT 286 )<br />
Panel A<br />
Panel B<br />
Panel C<br />
Product Size Cat.# Price ($)<br />
Anti-ACTIVE ® CaM KII pAb, Rabbit, (pT 286 ) 40 μl V1111 678<br />
For Research Use Only. Not for Use in Diagnostic Procedures.<br />
Description: This polyclonal antibody (pAb) is specific for the multifunctional<br />
calcium/calmodulin-dependent protein kinase CaM kinase II (CaM KII) that is<br />
phosphorylated on threonine 286 (pT 286 ). The Anti-ACTIVE ® CaM KII pAb was<br />
raised against the phosphothreonine-containing peptide.<br />
Features:<br />
• Specificity: Preferentially detects CaM KII autophosphorylated on<br />
threonine 286.<br />
• Immunogen: Threonine-phosphorylated peptide corresponding to the<br />
phosphorylated Thr 286 region of the mammalian calcium/calmodulindependent<br />
protein kinase.<br />
• Antibody Form: Affinity-purified rabbit IgG; supplied in 10mM sodium<br />
phosphate (pH 7.4), 20mM NaCl.<br />
• Value: When used at the recommended dilution of 1:5,000, it generates<br />
up to 200ml of blotting solution sufficient for 20 Western blots of 10ml<br />
each.<br />
Storage Conditions: Store at –20°C.<br />
Protocol<br />
Anti-ACTIVE ® CaM KII pAb, (pT 286 ) and Anti-ACTIVE ® Qualified<br />
Secondary Antibody Conjugate Technical Bulletin<br />
kDa<br />
250 –<br />
98 –<br />
64 –<br />
50 –<br />
36 –<br />
30 –<br />
16 –<br />
6 –<br />
4 –<br />
0.5µg 2.5µg 5µg 12.5µg 25µg<br />
+ – + – + – + – + –<br />
1 2 3 4 5 6 7 8 9 10 11<br />
– Phospho<br />
T 286<br />
CaM KII<br />
2055TA02_8A<br />
Part#<br />
TB264<br />
Immunocytochemical detection of autophosphorylated CaM KII in<br />
PC12 cells with Anti-ACTIVE ® CaM KII pAb. PC12 cells were adhered<br />
to slides coated with collagen, fixed in 10% paraformaldehyde for<br />
30 minutes, rinsed in PBS and permeabilized in methanol for 10 minutes<br />
at –20˚C. The cells were then blocked in 1% BSA in PBS for 45 minutes<br />
followed by 2% horse serum in PBS for 60 minutes. Cells were incubated<br />
overnight at 4˚C with (Panel A) Ab alone, (Panel B) Ab preincubated<br />
with a nonphosphorylated CaM KII peptide fragment (1µg/ml) or<br />
(Panel C) Ab preincubated with a phosphorylated CaM KII peptide fragment<br />
(1µg/ml). The Anti-ACTIVE ® CaM KII pAb was used at 1:500 dilution and<br />
preincubated with peptide for 8 hours at 4˚C. After incubation with the<br />
Anti-ACTIVE ® CaM KII pAb or Ab/peptide mixture, the cells were rinsed in<br />
PBS and incubated with a donkey anti-rabbit FITC-conjugated secondary<br />
Ab (1:500) for 60 minutes at room temperature. Staining was visualized<br />
with a Zeiss ® fluorescence microscope. The results demonstrate that<br />
preincubation of the Anti-ACTIVE ® CaM KII pAb with phosphorylated<br />
CaM KII peptide completely abolishes immunostaining (Panel C), but<br />
preincubation with nonphosphorylated CaM KII peptide has no effect on<br />
immunostaining (Panel A versus Panel B). Protocols developed and<br />
performed at <strong>Promega</strong>.<br />
2057TA02_8A<br />
10<br />
Imaging and Immunological Detection<br />
Detection of CaM KII by Anti-ACTIVE ® CaM KII pAb in Western<br />
analysis of rat brain homogenate. Lanes 2, 4, 6, 8 and 10 contain<br />
autophosphorylated (+) brain cytosolic protein in the amounts shown; lanes<br />
3, 5, 7, 9 and 11 contain nonphosphorylated (–) brain cytosolic protein in<br />
the amounts shown. The presence of the autophosphorylated CaM KII was<br />
detected using the Anti-ACTIVE ® CaM KII pAb diluted 1:5,000.<br />
Section<br />
Contents<br />
For complete and up-to-date product information visit: www.promega.com/catalog<br />
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Table of<br />
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