702084

antibody from Invitrogen Antibodies

Targeting: TRIM28 KAP1, PPP1R157, RNF96, TF1B, TIF1B

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Flow cytometry (Supportive data in Antibodypedia)

Antibody Data

Product number

702084

Provider

Invitrogen Antibodies

Product name

Phospho-TRIM28 (Ser824) Recombinant Rabbit Monoclonal Antibody (6H11L46)

Antibody type

Monoclonal

Antigen

Synthetic peptide

Reactivity

Human

Host

Rabbit

Isotype

IgG

Antibody clone number

6H11L46

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 PC-3 (Lane 1), PC-3 treated with Neocarzinostatin (200 ng/mL for 30 min) (Lane 2), Neuro-2a (Lane 3), Neuro-2a treated with Neocarzinostatin (200 ng/mL for 30 min) (Lane 4). The blots were probed with Anti-Phospho-TRIM28 (Ser824) Recombinant Rabbit Monoclonal Antibody (Product # 702084, 1 µg/mL). A 89 kDa band corresponding to Phospho-TRIM28 (Ser824), that specifically increases upon Neocarzinostatin treatment is as shown. The blots were detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:5000 dilution). 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, HeLa cells were fixed and permeabilized for detection of endogenous KAP1 pS824 using Anti-Phospho-TRIM28 (Ser824) Recombinant Rabbit Monoclonal Antibody (Product # 702084, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Detection and localization of Phospho-TRIM28 (Ser824) (green) in the nucleus can be clearly observed in cells treated with Neocarzinostatin (200 ng/mL, 15 min) as compared to untreated cells. Antibody specificity was demonstrated by competition with the Phospho-TRIM28 (Ser824) peptide, which results in loss of signal. No competition was observed with the non-phospho peptide. The images were captured at 60X magnification.

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Flow Cytometry analysis of endogenous Phospho-TRIM28 (Ser824) was performed on HeLa cells (untreated) and HeLa cells treated with 200 ng/mL Neocarzinostatin for 30 minutes. Cells were labeled with Anti-Phospho-TRIM (Ser824) Recombinant Rabbit Monoclonal Antibody (Product # 702084, 5 µg/ 1M cells) and detected with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, (Alexa Fluor® 488 conjugate, Product # A27034, 0.4 µg/mL, 1:2500). Rabbit IgG was used as the isotype control to determine nonspecific binding. A representative of 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer (Product # 4468770).