- PA5-17812 - Invitrogen Antibodies SYK antibody

Submitted references Molecular basis of carrageenan-induced cytokines production in macrophages.

Lopes AH, Silva RL, Fonseca MD, Gomes FI, Maganin AG, Ribeiro LS, Marques LMM, Cunha FQ, Alves-Filho JC, Zamboni DS, Lopes NP, Franklin BS, Gombault A, Ramalho FS, Quesniaux VFJ, Couillin I, Ryffel B, Cunha TM
Cell communication and signaling : CCS 2020 Sep 7;18(1):141

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of Syk was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR819133_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of Syk was performed by loading 30 µg of A-431 wild type (Lane 1), A-431 Cas9 (Lane 2) and A-431 Syk KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-Syk Polyclonal Antibody (Product # PA5-17812, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to Syk.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-Syk Polyclonal Antibody (Product # PA5-17812) and a 72kDa band corresponding to Tyrosine-protein kinase SYK was observed across cell lines tested. Whole cell extracts (30 µg lysate) of Ramos (Lane 1), RAW 264.7 (Lane 2), A-431 (Lane 3), NIH:OVCAR-3 (Lane 4), Jurkat (Lane 5), THP-1 (Lane 6) and Daudi (Lane 7) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Tyrosine-protein kinase SYK was performed using 70% confluent log phase Ramos cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Syk Polyclonal Antibody (Product # PA5-17812) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing cytoplasmic as well as membrane localization. Panel e represents low or negative expression in Jurkat. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 2 Role of TRIF/Syk/reactive oxygen species on Cg-induced Il1b mRNA. Peritoneal macrophages were stimulated with Cg (300 mug/ml) or medium. a ROS production were quantified in supernatant by the fluorescent probe assay (H2DCFDA) after indicated times. b, c The supernatant were collect after pre-treated cells with N-acetylcysteine (NAC - 3 mM) to quantify Il1b mRNA gene expression by qPCR and IL-1beta production by Elisa after Cg (6 h). d Quantification of ROS production by (H2DCFDA probe) in cells from naive WT vs deficient for Trif -/- after Cg (3 h). e, f Pre-treated cells with selective inhibitor of Syk (iSyk 1, 3 muM) and stimulated with Cg (6 h) to quantify IL-1beta by ELISA and Il1b mRNA gene expression induced by Cg (3 h), medium or (iSyk 3 muM). g Quantification of ROS production (H2DCFDA probe) stimulated with Cg and iSyk (3 uM) after 3 h. h Western blotting analysis of (pSyk) expression compared cells from WT vs Trif -/- . Data are represent the mean +- SD of four independent experiments compared Control vs WT (Cg) or WT vs Knockout/Treatments groups to determine the level of statistical significance (* p < 0.05; ** p < 0.01; and *** p < 0.001; ns, not significant)

PA5-17812

Antibody data