CST Guide:

Section III: Workflow Tools
chapter 18: Investigation
Treatment with
Calyculin A induces
a broad increase in
phosphorylation at
phospho-Ser 14-3-3
substrate motif sites.
How CST motif and PTM-specific
antibodies can benefit your research:
• Multi-pathway Analysis: motif antibodies allow the detection of a broad range of substrates for
a single kinase, so multiple pathways can be analyzed simultaneously using a single antibody.
SDS-PAGE
→
• Multiplex Applications: many CST motif antibodies are validated for IP, peptide ELISA (E-P),
and/or immunohistochemistry (IHC) in addition to WB, enabling flexibility in experimental design
and the possibility of confirming results via different applications.
• Proteomic Analysis: CST PTMScan ® services and kits integrate motif and PTM-specific
antibodies with mass spectrometry (MS) to enable the discovery and analysis of hundreds
to thousands of PTM sites using standard LC-MS techniques.
Untreated
→
IPGE
Calyculin A
→
IPGE
Phospho-(Ser) 14-3-3 Binding
Motif Antibody #9601: WB analysis of
extracts from Jurkat cells, untreated or
Calyculin A-treated (0.1 µM for 30 min),
using #9601. Proteins were separated
by 2-D electrophoresis prior to blotting.
#9646 Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb
IP †
RXX(S*/T*)
(Sepharose ® Bead Conjugate)
#9611 Phospho-(Ser/Thr) Akt Substrate Antibody W, IP † , IHC-P, E-P (K/R)XX(S*/T*)
#10001 Phospho-Akt Substrate (RXRXXS*/T*) (23C8D2) Rabbit mAb W, E-P RXRXX(S*/T*)
#5759 Phospho-(Ser/Thr) AMPK Substrate (P-S/T 2 -102) Rabbit mAb W, IP † , E-P (L/M)XRXX(S*/T*),
RXX(S*/T*)
#2909 Phospho-(Ser/Thr) ATM/ATR Substrate (4F7) Rabbit mAb W L(S*/T*)Q
#2851 Phospho-(Ser/Thr) ATM/ATR Substrate Antibody W, IP † , IHC-P, E-P L(S*/T*)Q
#6966 Phospho-(Ser/Thr) ATM/ATR Substrate (S*/T*QG) (P-S/T2-100) Rabbit mAb W, IP † (S*/T*)QG, (S*/T*)Q
#8738 Phospho-(Ser/Thr) CK2 Substrate (P-S/T 3 -100) Rabbit mAb W (S*/T*)DXE
#9634 Phospho-(Ser/Thr) PDK1 Docking Motif (18A2) Mouse mAb W, IP † , E-P (F/K)XX(F/Y)(S*/T*)(F/Y)
#9631 Phospho-(Ser/Thr) Phe Antibody W, IP † , E-P (F/Y/W)(S*/T*)(F)
#9624 Phospho-PKA Substrate (RRXS*/T*) (100G7E) Rabbit mAb W, IP † , E-P (K/R)(K/R)X(S*/T*)
#9621 Phospho-(Ser/Thr) PKA Substrate Antibody W, IP † , IHC-P, E-P (K/R)(K/R)X(S*/T*)
#4381 Phospho-(Ser/Thr) PKD Substrate Antibody W, E-P LXRXX(S*/T*)
Phospho-Tyrosine Application Motif Recognized
#8954 Phospho-Tyrosine (P-Tyr-1000) Rabbit mAb W, IP † , IF-IC, F Y*
#9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100) W, IP † , IHC-P, IF-P, Y*
IF-F, IF-IC, F, E-P
#9414 Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Alexa Fluor ® 488 Conjugate) F Y*
#9415 Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Alexa Fluor ® 647 Conjugate) IF-IC, F Y*
#9417 Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Biotinylated) W, IP † , IHC-P Y*
Antibody recognition
of the PKA substrate
motif is phosphorylation-dependent.
+ + + +
– – – + – – – –
Phospho-(Ser/Thr) PKA Substrate
Antibody #9621: WB analysis of extracts
from A-431 cells, phosphorylated in vitro
by protein kinase A (A), Erk2 (B) or cdc2/
cyclin A (C), plus or minus PKA inhibitor
(PKI), using #9621.
Lanes
1. PKA
2. Erk2
3. cdc2/Cyclin A
A B C
Cell Extract
PKI
#5465 Phospho-Tyrosine Mouse mAb (P-Tyr-100) (HRP Conjugate) W, E-P Y*
Motif and PTM-specific antibodies available from CST
As with our more traditional antibodies, CST’s motif and PTM antibody development team invests significant
time and resources ensuring that these reagents are highly specific for the motif or PTM of interest.
These antibodies are validated by a number of methods including WB, peptide arrays, and peptide IP
followed by LC-MS analysis to confirm that the antibody performs to the highest possible standard.
#8095 Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Magnetic Bead Conjugate) IP † Y*
#9419 Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Sepharose ® Bead Conjugate) IP † Y*
#9416 Phospho-Tyrosine Mouse mAb (P-Tyr-102) W, IP † , IHC-P, F, E-P Y*
Nitro-Tyrosine Application Motif Recognized
#9691 Nitro-Tyrosine Antibody W Y-NO2
EGF stimulates
phosphorylation of
tyrosine residues in
many cellular proteins.
Phospho-Serine Application Motif Recognized
#9615 Phospho-(Ser) Kinase Substrate Antibody Sampler Kit
#9606 Phospho-(Ser) 14-3-3 Binding Motif (4E2) Mouse mAb W, IP † , E-P (K/R)XXS*P
#9601 Phospho-(Ser) 14-3-3 Binding Motif Antibody W, IP † , IHC-P, E-P (K/R)XXS*P
#9607 Phospho-ATM/ATR Substrate (S*Q) (D23H2/D69H5) Rabbit mAb W, IP † S*Q
#6967 Phospho-(Ser) PKC Substrate (P-S 3 -101) Rabbit mAb W, IP † , E-P (K/R)XS*X(K/R)
Acetylated Lysine Application Motif Recognized
#9814 Acetylated-Lysine (Ac-K 2 -100) Rabbit mAb W, IP † , ChIP, E-P Ac-K
#6952 Acetylated-Lysine (Ac-K 2 -100) Rabbit mAb (HRP Conjugate) W Ac-K
#9681 Acetylated-Lysine Mouse mAb (Ac-K-103) W, E-P Ac-K
#9441 Acetylated-Lysine Antibody W, IP † , IHC-P, IF-IC, Ac-K
ChIP, E-P
kDa
200
140
100
80
#2325 Phospho-MAPK/CDK Substrates (PXS*P or S*PXR/K) (34B2) Rabbit mAb W, IP † , IHC-P, E-P PXS*P, S*PX(K/R)
#5501 Phospho-MAPK/CDK Substrates (PXS*P or S*PXR/K) (34B2) Rabbit mAb IP †
PXS*P, S*PX(R/K)
(Sepharose ® Bead Conjugate)
#9477 Phospho-(Ser) CDKs Substrate (P-S 2 -100) Rabbit mAb W, IP † , E-P (K/H)S*P
#2261 Phospho-(Ser) PKC Substrate Antibody W, IP † , E-P (K/R)XS*(Hyd)(K/R)
#2981 Phospho-(Ser) Arg-X-Tyr/Phe-X-pSer Motif Antibody W, IP † , E-P RX(F/Y)XS*
Phospho-Threonine Application Motif Recognized
#9391 Phospho-Threonine-Proline Mouse mAb (P-Thr-Pro-101) W, IHC-P, E-P T*P
#5243 Phospho-PLK Binding Motif (ST*P) (D73F6) Rabbit mAb W, IP † , E-P ST*P
#2351 Phospho-Threonine-X-Arginine Antibody W, IHC-P, E-P T*X(K/R)
#3004 Phospho-Thr-Pro-Glu (C32G12) Rabbit mAb W, IP † , E-P T*PE, T*P
#9386 Phospho-Threonine (42H4) Mouse mAb W, IP † , E-P T*
#9381 Phospho-Threonine Antibody (P-Thr-Polyclonal) W, IP † , E-P T*
#8781 Phospho-Threonine Antibody (P-Thr-Polyclonal)
IP † T*
(Sepharose Bead Conjugate)
#6949 Phospho-Threonine Antibody (P-Thr-Polyclonal) (HRP Conjugate) W, E-P T*
Mono-/Di-Methyl-Arginine Application Motif Recognized
#8015 Mono-Methyl Arginine (Me-R 4 -100) Rabbit mAb W, IP † , E-P Me-R
#8711 Mono-Methyl Arginine (R*GG) (D5A12) Rabbit mAb W, IP † , E-P Me-RGG
#13522 Asymmetric Di-Methyl Arginine Motif [adme-R] Rabbit mAb W ADMe-R
#13222 Symmetric Di-Methyl Arginine Motif [sdme-RG] Rabbit mAb W SDMe-RG
Ubiquitin Linkage Application Motif Recognized
#8081 K48-Linkage Specific Polyubiquitin (D9D5) Rabbit mAb W Polyubiquitin formed
by K48 linkage
#12805 K48-linkage Specific Polyubiquitin (D9D5) Rabbit mAb (HRP Conjugate) W Polyubiquitin formed
by K48 linkage
#4289 K48-linkage Specific Polyubiquitin Antibody W Polyubiquitin formed
by K48 linkage
#5621 K63-Linkage Specific Polyubiquitin (D7A11) Rabbit mAb W Polyubiquitin formed
by K63 linkage
#12930 K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb (HRP Conjugate) W Polyubiquitin formed
by K63 linkage
60
50
40
30
– + hEGF
Phospho-Tyrosine (P-Tyr-1000) Rabbit
mAb #8954: WB analysis of extracts from
A-431 cells, untreated or treated with
Human Epidermal Growth Factor (hEGF)
#8916 (100 ng/ml, 5 min), using #8954.
†
IP denotes suitability for standard IP, not for peptide immunoaffinity enrichment prior to LC-MS/MS.
Phospho-Serine/Threonine Application Motif Recognized
#9920 Phospho-(Ser/Thr) Kinase Substrate Antibody Sampler Kit
#9614 Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb W, IP † , IHC-P, E-P RXX(S*/T*)
#6950 Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb (HRP Conjugate) W RXX(S*/T*)
#8050 Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb
(Magnetic Bead Conjugate)
IP †
RXX(S*/T*)
PTMScan® Proteomics Services
Quantitative PTM profiling — see page 258 or visit our website. www.cellsignal.com/discovery
256 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys.
www.cellsignal.com/investigation
257