Recombinant Anti-CCT2 antibody [EPR4084] (ab92746)

Blocking/Diluting buffer: 5% NFDM/TBST

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue sections labeling CCT2 with purified ab92746 at 1/24000 dilution (0.012 µg/mL). Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 1 (pH 6.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue sections labeling CCT2 with purified ab92746 at 1/24000 dilution (0.012 µg/mL). Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 1 (pH 6.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells, labeling CCT2 with Purified ab92746 at 1/30 dilution (10µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue). 

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue sections labeling CCT2 with purified ab92746 at 1/24000 dilution (0.012 µg/mL). Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 1 (pH 6.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Immunocytochemistry/Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling CCT2 with Purified ab92746 at 1:500 dilution (0.6 µg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Purified ab92746 at 1:30 dilution (2μg) immunoprecipitating CCT2 in HeLa whole cell lysate.
Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg
Lane 2 (+): ab92746 + HeLa whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab92746 in HeLa whole cell lysate.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 57 kDa

ab92746, unpurified, at a 1/100 dilution, staining CCT2 in formalin fixed, paraffin embedded (1) Human colon tissue and (2) Human kidney tissue by Immunohistochemistry. Detection: DAB staining.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

ab92746, unpurified, at a 1/100 dilution, staining CCT2 in HeLa cells by Immunofluorescence.

Overlay histogram showing HeLa cells stained with ab92746, unpurified, (red line), unpurified. The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab92746, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.