44-233G

antibody from Invitrogen Antibodies

Targeting: SYK

Western blot (Supportive data in Antibodypedia)

Antibody Data

Product number

44-233G

Provider

Invitrogen Antibodies

Product name

Phospho-Syk (Tyr317) Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human, Mouse

Host

Rabbit

Isotype

IgG

Vial size

100 µL

Storage

-20°C

References

Submitted references

An Allosteric Shift in CD11c Affinity Activates a Proatherogenic State in Arrested Intermediate Monocytes.

Hernandez AA, Foster GA, Soderberg SR, Fernandez A, Reynolds MB, Orser MK, Bailey KA, Rogers JH, Singh GD, Wu H, Passerini AG, Simon SI
Journal of immunology (Baltimore, Md. : 1950) 2020 Nov 15;205(10):2806-2820

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Peptide Competition and Phosphatase Treatment. Lysates prepared from Jurkat cells left unstimulated (1) or stimulated with H2O2 (2-6) and mouse OKT-5 lymphocytes left unstimulated (7) or stimulated with H2O2 (8) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (Lanes 1-5, 7 & 8) or treated with L-phosphatase (6), blocked with a 3% Milk-TBST buffer for one hour at room temperature, and incubated with Syk (pY317) antibody for two hours at room temperature in a 3% Milk-TBST buffer, following prior incubation with: no peptide (1, 2, 6), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphotyrosine-containing peptide (4), or, the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F (ab’)2 anti-rabbit IgG HRP conjugate (Product # ALI4404) and bands were detected using the Pierce SuperSignal™ method. The data show that the signal was induced upon H2O2 treatment. The data also show peptide corresponding to Syk (pY317) blocks the antibody signal, and that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Peptide Competition and Phosphatase Treatment. Lysates prepared from Jurkat cells left unstimulated (1) or stimulated with H2O2 (2-6) and mouse OKT-5 lymphocytes left unstimulated (7) or stimulated with H2O2 (8) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (Lanes 1-5, 7 & 8) or treated with L-phosphatase (6), blocked with a 3% Milk-TBST buffer for one hour at room temperature, and incubated with Syk (pY317) antibody for two hours at room temperature in a 3% Milk-TBST buffer, following prior incubation with: no peptide (1, 2, 6), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphotyrosine-containing peptide (4), or, the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F (ab’)2 anti-rabbit IgG HRP conjugate (Product # ALI4404) and bands were detected using the Pierce SuperSignal™ method. The data show that the signal was induced upon H2O2 treatment. The data also show peptide corresponding to Syk (pY317) blocks the antibody signal, and that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.