Revision 3

#44170

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Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Applications:
W, W-S, IP, FC-FP

Reactivity:
H M R

Sensitivity:
Endogenous

MW (kDa):
72

Source/Isotype:
Rabbit IgG

UniProt ID:
#P43405

Entrez-Gene Id:
6850

Product Usage Information

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:10 - 1:50
Immunoprecipitation 1:200
Flow Cytometry (Fixed/Permeabilized) 1:400 - 1:1600

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/mL BSA, 50% glycerol, and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity/Sensitivity

Phospho-Syk (Tyr323) (F9H7U) Rabbit Monoclonal Antibody recognizes endogenous levels of Syk protein only when phosphorylated at Tyr323. This antiody does not cross-react with other tyrosine-phosphorylated members of the Syk/Zap-70 tyrosine kinase family. Species reactivity for flow cytometry is human only.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr323 of human Syk protein.

Background

Syk is a protein tyrosine kinase that plays an important role in intracellular signal transduction in hematopoietic cells (1-3). Syk interacts with immunoreceptor tyrosine-based activation motifs (ITAMs) located in the cytoplasmic domains of immune receptors (4). It couples the activated immunoreceptors to downstream signaling events that mediate diverse cellular responses, including proliferation, differentiation, and phagocytosis (4). There is also evidence of a role for Syk in nonimmune cells and investigators have indicated that Syk is a potential tumor suppressor in human breast carcinomas (5). Tyr323 is a negative regulatory phosphorylation site within the SH2-kinase linker region in Syk. Phosphorylation at Tyr323 provides a direct binding site for the TKB domain of Cbl (6,7). Tyr352 of Syk is involved in the association of PLCγ1 (8). Tyr525 and Tyr526 are located in the activation loop of the Syk kinase domain; phosphorylation at Tyr525/526 of human Syk (equivalent to Tyr519/520 of mouse Syk) is essential for Syk function (9).

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

W: Western Blotting W-S: Simple Western™ IP: Immunoprecipitation FC-FP: Flow Cytometry (Fixed/Permeabilized)

Cross-Reactivity Key

H: Human M: Mouse R: Rat

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 3

Cell Signaling Technology Logo
Western blot analysis of extracts from Ramos cells, untreated (-) or treated with anti-human IgM (12 µg/mL, 5 min; +), using Phospho-Syk (Tyr323) (F9H7U) Rabbit mAb (upper), Phospho-Syk (Tyr323) Antibody #2715 (upper-middle), Syk (D3Z1E) XP® Rabbit mAb #13198 (lower-middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Western Blotting Image 1: Phospho-Syk (Tyr323) (F9H7U) Rabbit Monoclonal Antibody
Western blot analysis of extracts from Ramos, RAW 264.7, and YB2/0 cells, untreated (-) or treated with hydrogen peroxide (H2O2) (11 mM, 1 min; +), using Phospho-Syk (Tyr323) (F9H7U) Rabbit mAb (upper), Syk (D3Z1E) XP® Rabbit mAb #13198 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Western Blotting Image 2: Phospho-Syk (Tyr323) (F9H7U) Rabbit Monoclonal Antibody
Simple Western™ analysis of lysates (0.8 mg/mL) from serum-starved Ramos cells treated with hydrogen peroxide (H2O2; 10uM, 2 minutes) using Phospho-Syk (Tyr323) (F9H7U) Rabbit mAb #44170. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Western Blotting Image 1: Phospho-Syk (Tyr323) (F9H7U) Rabbit Monoclonal Antibody
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 3

Cell Signaling Technology Logo
Immunoprecipitation of phospho-Syk (Tyr323) protein from RAW 264.7 cell extracts. Cells were treated with hydrogen peroxide (11 mM, 1 min). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-Syk (Tyr323) (F9H7U) Rabbit mAb. Western blot analysis was performed using Syk (D3Z1E) XP® Rabbit mAb #13198. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.
Immunoprecipitation Image 1: Phospho-Syk (Tyr323) (F9H7U) Rabbit Monoclonal Antibody
Flow cytometric analysis of untreated Ramos cells (blue) and Ramos cells treated with anti-human IgM (12 µg/mL, 5 min; green) using Phospho-Syk (Tyr323) (F9H7U) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Flow Cytometry Image 1: Phospho-Syk (Tyr323) (F9H7U) Rabbit Monoclonal Antibody
Flow cytometric analysis of Ramos cells treated with anti-human IgM (10 µg/mL, 30 min), and either treated post-fixation with λ phosphatase (blue) or untreated (green), using Phospho-Syk (Tyr323) (F9H7U) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Flow Cytometry Image 2: Phospho-Syk (Tyr323) (F9H7U) Rabbit Monoclonal Antibody
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.