44-969

antibody from Invitrogen Antibodies

Targeting: PRKCH PKC-L, PKCL, PRKCL

Western blot (Supportive data in Antibodypedia)

Flow cytometry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

44-969

Provider

Invitrogen Antibodies

Product name

Phospho-PKC eta (Thr655) Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human

Host

Rabbit

Isotype

IgG

Vial size

100 µL

Storage

-20°C

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Peptide Competition and Phosphatase Treatment. Lysates prepared from Jurkat cells stimulated with PMA were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-9) or treated with lambda phosphatase (10), blocked with a 5% BSA-TBST buffer overnight at 4°C, and incubated with 1:2000 dilution of Phospho-PKCeta (Thr655) antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 10), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine containing peptide (3), the phosphopeptide immunogen (4), or, the phosphopeptide corresponding to the immunogen from other PKC isoforms (5-9). After washing, membranes were incubated with goat F (ab and quote;)2 anti-rabbit IgG HRP conjugate (Product # ALI4404) and bands were detected using the Pierce SuperSignal™ method. The data show that the peptide corresponding to PKCeta (pT655) blocks the antibody signal. The antibody signal was not blocked by the peptides corresponding to PKC isoforms phospho-alpha (Thr638), phospho-betaI (Thr642), phospho-betaI (ThrT641), phospho-gamma (Th655) and zeta (Thr560), thereby demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Flow cytometry analysis of PKC-eta [pT655] was done on HeLa cells treated with PMA (200nM, 20 minutes). Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with PKC-eta [pT655] Rabbit Polyclonal Antibody (44969, red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Antibody specificity is demonstrated by Peptide Array. Western blotting of Phospho-PKC eta (Thr655) using anti-Phospho-PKC eta (Thr655) Polyclonal Antibody (Product # 44-969) shows loss of signal with the specific peptide and not with other relevant peptides.