Anti-PKC theta/PRKCQ antibody [27/PKC theta] - BSA and Azide Free

• IP

Supplier Data

Immunoprecipitation - Anti-PKC theta/PRKCQ antibody [27/PKC theta] - BSA and Azide Free (AB302892)

This data was developed using ab302891, the same antibody clone in a different buffer formulation. PKC theta/PRKCQ was immunoprecipitated from 0.35 mg K-562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate 10 µg with ab302891 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab302891 at 1/1000 dilution. Peroxidase-Conjµated Goat anti-Mouse IgG (H+L) was used at 1/10000 dilution. Lane 1 : K-562 whole cell lysate 10 µg Lane 2 : ab302891 IP in K-562 whole cell lysate Lane 3 : Mouse IgG2a, kappa monoclonal isotype control (ab18413) instead of ab302891 in K-562 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 3 seconds.

All lanes:

Immunoprecipitation - Anti-PKC theta/PRKCQ antibody [27/PKC theta] (<a href='/en-us/products/primary-antibodies/pkc-theta-prkcq-antibody-27-pkc-theta-ab302891'>ab302891</a>) at 1/30 dilution

All lanes:

K-562 (human chronic myelogenous leukemia lymphoblast)

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Observed band size: 82 kDa

false

Exposure time: 3s

• WB

Supplier Data

Western blot - Anti-PKC theta/PRKCQ antibody [27/PKC theta] - BSA and Azide Free (AB302892)

This data was developed using ab302891, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST. The blot of lane 4 was developed using a high sensitivity ECL substrate. Exposure time : Lane 1-3 : 92 seconds; Lane 4 : 59 seconds.

All lanes:

Western blot - Anti-PKC theta/PRKCQ antibody [27/PKC theta] (<a href='/en-us/products/primary-antibodies/pkc-theta-prkcq-antibody-27-pkc-theta-ab302891'>ab302891</a>) at 1/1000 dilution

Lane 1:

Mouse spleen tissue lysate at 20 µg

Lane 2:

Mouse thymus tissue lysate at 20 µg

Lane 3:

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

Lane 4:

Human cerebellum tissue lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) (ZB-2305) at 1/10000 dilution

Observed band size: 81 kDa

true

• WB

Supplier Data

Western blot - Anti-PKC theta/PRKCQ antibody [27/PKC theta] - BSA and Azide Free (AB302892)

This data was developed using ab302891, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST. Exposure time : 26 seconds.

All lanes:

Western blot - Anti-PKC theta/PRKCQ antibody [27/PKC theta] (<a href='/en-us/products/primary-antibodies/pkc-theta-prkcq-antibody-27-pkc-theta-ab302891'>ab302891</a>) at 1/1000 dilution

Lane 1:

Active human PKC beta 1 fl length protein 5 ng at 5 ng

Lane 2:

Active human PKC delta fl length protein 5 ng at 5 ng

Lane 3:

Active human PKC theta fl length protein 10 ng at 10 ng

Lane 4:

Active human PKC zeta fl length protein 10 ng at 10 ng

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) (ZB-2305) at 1/10000 dilution

Observed band size: 100 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-PKC theta/PRKCQ antibody [27/PKC theta] - BSA and Azide Free (AB302892)

This data was developed using ab302891, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST. Exposure time : 37 seconds.

All lanes:

Western blot - Anti-PKC theta/PRKCQ antibody [27/PKC theta] (<a href='/en-us/products/primary-antibodies/pkc-theta-prkcq-antibody-27-pkc-theta-ab302891'>ab302891</a>) at 1/1000 dilution

Lane 1:

Active human PKC alpha fl length protein 60 ng at 60 ng

Lane 2:

Active human PKC beta 2 fl length protein 5 ng at 5 ng

Lane 3:

Active human PKC epsilon fl length protein 50 ng at 50 ng

Lane 4:

Active human PKC theta fl length protein 5 ng at 5 ng

Lane 5:

Active human PKC mu fl length protein 50 ng at 50 EU/ng

Lane 6:

Active human PKC iota fl length protein 10 ng at 10 ng

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) (ZB-2305) at 1/10000 dilution

Observed band size: 100 kDa

false

Exposure time: 37s

• WB

Lab

Western blot - Anti-PKC theta/PRKCQ antibody [27/PKC theta] - BSA and Azide Free (AB302892)

This data was developed using ab302891, the same antibody clone in a different buffer formulation.

Western blot : Mouse Monoclonal[27/PKC theta] to PKC theta/PRKCQ ab302891 staining at 1/1000 dilution, shown in green; Rabbit anti GAPDH (ab181602) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 75 kDa in Wild-type A549 cell lysates with no signal observed at this size in PRKCQ knockout A549 cell line.

To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse 800CW & Goat anti-Rabbit 680RD at 1/20,000 dilution.

Lanes 1 - 4:

Western blot - Anti-PKC theta/PRKCQ antibody [27/PKC theta] (<a href='/en-us/products/primary-antibodies/pkc-theta-prkcq-antibody-27-pkc-theta-ab302891'>ab302891</a>) at 1/1000 dilution

Lanes 1 - 4:

Western blot - Anti-PKC theta/PRKCQ antibody [27/PKC theta] - BSA and Azide Free (ab302892) at 1/1000 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

PRKCQ knockout A549 at 20 µg

Lane 3:

Jurkat at 20 µg

Lane 4:

Raji at 20 µg

Secondary

All lanes:

Goat anti-Mouse 800CW & Goat anti-Rabbit 680RD at 1/20000 dilution

Predicted band size: 82 kDa

Observed band size: 75 kDa

false