Protocols and Applications Guide (US Letter Size) - Promega

||||||| 7Cell Signaling
non-phosphorylated
(+1 net charge)
colored peptide substrate
(+1 net charge)
Incubate peptide with
kinase-containing sample.
phosphorylated
(–1 net charge)
Separate phosphorylated and nonphosphorylated
forms by agarose gel electrophoresis.
Net Charge
of Peptide
+1
0
–1
cathode
anode
P =
Peptide Species
nonphosphorylated
P =
phosphorylated
Figure 7.12. Schematic diagram of the PepTag® Non-Radioactive
Protein Kinase Assay procedure.
Additional Resources for the PepTag® Non-Radioactive
Protein Kinase Assay
Technical Bulletins and Manuals
TB132 PepTag® Assay for Non-Radioactive Detection
of Protein Kinase C or cAMP-Dependent
Protein Kinase Technical Bulletin
(www.promega.com/tbs/tb132/tb132.html)
Promega Publications
BR095 Signal Transduction Resource
(www.promega.com
/guides/sigtrans_guide/default.htm)
Citations
DiPasquale, G. and Chiorini, J.A. (2004) PKA/PrKX activity
is a modulator of AAV/adenovirus interaction. EMBO J. 22,
1716–24.
The PepTag® Non-Radioactive cAMP-Dependent Protein
Kinase Assay was used to analyze PKA activity in HeLa
cells infected with a human parvovirus. Lysates were
prepared by sonication of cells in PBS. Data is presented
as either a percent of control or fold increase over control.
PubMed Number: 12660177
Protocols & Applications Guide
www.promega.com
rev. 3/09
0383MA02_2A
IV. Phosphorylation-Specific Antibodies
The Anti-ACTIVE® phosphorylation-specific antibodies
were developed to provide an accurate measure of enzyme
activation. These antibodies specifically recognize the
active, phosphorylated form of a given kinase. The
Anti-ACTIVE® Antibodies are raised against
phosphorylated peptide sequences present in the activating
loop of a number of protein kinases. Whether used in
Western analysis, immunocytochemistry or
immunohistochemical staining, the Anti-ACTIVE® MAPK,
JNK, p38 and CaM KII Antibodies will recognize only the
active form of the enzyme.
A. Phosphorylation-Specific Antibodies in MAPK Signaling
Pathways
Anti-ACTIVE® MAPK, pAb, Rabbit, (pTEpY)
This antibody is an affinity purified polyclonal antibody
that specifically recognizes the dually phosphorylated,
active form of MAPK. The antibody is raised against a
dually phosphorylated peptide sequence representing the
catalytic core of the active ERK enzyme and recognizes the
active forms of ERK1, ERK2 and ERK7.
Anti-ACTIVE® JNK pAb, Rabbit, (pTPpY)
Anti-ACTIVE® JNK pAb is an affinity purified polyclonal
antibody that recognizes the dually phosphorylated, active
form of cJun N-terminal protein Kinase (JNK).
Anti-ACTIVE® JNK pAb is raised against a dually
phosphorylated peptide sequence representing the catalytic
core of the active JNK enzyme. The antibody recognizes
the active forms of JNK1, JNK2, and JNK3 isoforms.
Anti-ACTIVE® p38 pAb, Rabbit, (pTGpY)
Anti-ACTIVE® p38 Ab, Rabbit, is an affinity purified
polyclonal antibody that recognizes the active form of p38
kinase. The Anti-ACTIVE® p38 pAb is raised against the
dually phosphorylated peptide sequence representing the
catalytic core of the active p38 enzyme. The Anti-ACTIVE®
p38 pAb recognizes the active forms of p38α, γ, and δ
isoforms.
Western Blot Analysis with Anti-ACTIVE® MAPK, JNK
and p38 pAbs
Materials Required:
• Anti-ACTIVE® MAPK (Cat.# V8031), JNK (Cat.# V7931),
or p38 (Cat.# V1211) pAb
• Anti-ACTIVE® Qualified Donkey Anti-Rabbit IgG
(H+L), HRP (Cat.# V7951) or Donkey Anti-Rabbit IgG
(H+L) AP (Cat.# V7971) Secondary Antibodies
• protein sample transfered to nitrocellulose or PVDF
membrane
• bovine serum albumin, 1%
• TBS buffer
• TBST or PVDF buffer
• shaking platform
PROTOCOLS & APPLICATIONS GUIDE 7-11