Anti-PKA R2/PKR2 antibody [Y116] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-PKA R2/PKR2 antibody [Y116] - BSA and Azide free (AB247265)

This data was developed using ab32514, the same antibody clone in a different buffer formulation. Intracellular Flow Cytometry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling PKA R2/PKR2 with purified ab32514 at 1/20 dilution (10 μg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 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).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PKA R2/PKR2 antibody [Y116] - BSA and Azide free (AB247265)

This data was developed using ab32514, the same antibody clone in a different buffer formulation.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon cancer tissue sections labeling PKA R2/PKR2 with purified ab32514 at 1/100 dilution (0.84 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). 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.

• IP

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Immunoprecipitation - Anti-PKA R2/PKR2 antibody [Y116] - BSA and Azide free (AB247265)

This data was developed using ab32514, the same antibody clone in a different buffer formulation.
Purified ab32514 at 1/20 dilution (0.5μg) immunoprecipitating PKA R2/PKR2 in K-562 whole cell lysate.
Lane 1 (input) : K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate 10μg
Lane 2 (+) : ab32514 + K-562 whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab32514 in K-562 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 50 kDa

All lanes:

Immunoprecipitation - Anti-PKA R2/PKR2 antibody [Y116] (<a href='/en-us/products/primary-antibodies/pka-r2-pkr2-antibody-y116-ab32514'>ab32514</a>)

Predicted band size: 45 kDa

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• WB

Lab

Western blot - Anti-PKA R2/PKR2 antibody [Y116] - BSA and Azide free (AB247265)

This data was developed using the same antibody clone in a different buffer formulation (ab32514).

Lanes 1-3 : Merged signal (red and green). Green - ab32514 observed at 50 kDa. Red - loading control ab8245 observed at 36 kDa.

Unpurified ab32514 Anti-PKA R2/PKR2 antibody [Y116] was shown to specifically react with PKA R2/PKR2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265748 (knockout cell lysate ab257607) was used. Wild-type and PKA R2/PKR2 knockout samples were subjected to SDS-PAGE. ab32514 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PKA R2/PKR2 antibody [Y116] (<a href='/en-us/products/primary-antibodies/pka-r2-pkr2-antibody-y116-ab32514'>ab32514</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

PRKAR2A knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human PRKAR2A (PKA R2/PKR2) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-prkar2a-pka-r2-pkr2-knockout-hela-cell-line-ab265748'>ab265748</a>)

Lane 3:

K-562 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 45 kDa

Observed band size: 50 kDa

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• WB

Unknown

Western blot - Anti-PKA R2/PKR2 antibody [Y116] - BSA and Azide free (AB247265)

This data was developed using ab32514, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-PKA R2/PKR2 antibody [Y116] (<a href='/en-us/products/primary-antibodies/pka-r2-pkr2-antibody-y116-ab32514'>ab32514</a>) at 1/10000 dilution

Lane 1:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 45 kDa

Observed band size: 50 kDa

false