711415

antibody from Invitrogen Antibodies

Targeting: SYK

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

711415

Provider

Invitrogen Antibodies

Product name

Phospho-Syk (Tyr323, Tyr317) Recombinant Polyclonal Antibody (6HCLC)

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human

Host

Rabbit

Isotype

IgG

Antibody clone number

6HCLC

Vial size

100 µg

Concentration

0.5 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on Whole cell extracts (30 µg lysate) of U-937 (Lane 1), HEL 92.1.7 (Lane 2) and HEL 92.1.7 treated with Parvanadate (2mM for 5min) (Lane 3). The blots were probed with Anti-Syk (pY317-323) Recombinant Rabbit Polyclonal Antibody (Product # 711415, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 72 kDa band corresponding to Syk (pY317-323) was observed. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

For immunofluorescence analysis, Ramos cells were fixed and permeabilized for detection of Syk pY317 pY323 using Anti- Syk pY317 pY323 Recombinant Rabbit Monoclonal Antibody (Product # 711415, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of Syk pY317 pY323 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating membranous localization of Syk pY317 pY323. Panel e) shows loss of signal by competition with the Syk pY317 pY323 phospho peptide, demonstrating antibody specificity, and panel f) demonstrates no competition with the non-phosphorylated peptide. Panel g) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.