Anti-PACT (PKR activating protein) / PRKRA antibody

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PACT (PKR activating protein) / PRKRA antibody (AB31967)

ab31967 staining PACT (PKR activating protein) / PRKRA in Hek293 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab31967 at 5µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in green) and ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• WB

Project1883****

Western blot - Anti-PACT (PKR activating protein) / PRKRA antibody (AB31967)

All lanes:

Western blot - Anti-PACT (PKR activating protein) / PRKRA antibody (ab31967) at 1 µg/mL

Lane 1:

Testis (Mouse) Tissue Lysate - normal tissue at 10 µg

Lane 2:

PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 34 kDa

Observed band size: 34 kDa,55 kDa

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• IP

Unknown

Immunoprecipitation - Anti-PACT (PKR activating protein) / PRKRA antibody (AB31967)

PACT (PKR activating protein) / PRKRA was immunoprecipitated using 0.5mg Mouse Testis tissue, 5µg of Rabbit polyclonal to PACT (PKR activating protein) / PRKRA and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Mouse Testis tissue lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab31967.
Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band : 34kDa; PACT (PKR activating protein) / PRKRA

All lanes:

Immunoprecipitation - Anti-PACT (PKR activating protein) / PRKRA antibody (ab31967)

Predicted band size: 34 kDa

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• WB

Project1883****

Western blot - Anti-PACT (PKR activating protein) / PRKRA antibody (AB31967)

All lanes:

Western blot - Anti-PACT (PKR activating protein) / PRKRA antibody (ab31967) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg with Human PACT (PKR activating protein) / PRKRA peptide (<a href='/en-us/products/unavailable/human-pact-pkr-activating-protein-prkra-peptide-ab30768'>ab30768</a>)

Secondary

All lanes:

IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/15000 dilution

Predicted band size: 34 kDa

Observed band size: 34 kDa,60 kDa

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• WB

Lab

Western blot - Anti-PACT (PKR activating protein) / PRKRA antibody (AB31967)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : PACT (PKR activating protein)/PRKRA knockout HAP1 cell lysate (20 μg)
Lane 3 : K562 cell lysate (20 μg)
Lane 4 : HepG2 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab31967 observed at 36 kDa. Red - loading control, ab18058, observed at 124 kDa.

ab31967 was shown to recognize PACT (PKR activating protein)/PRKRA when PACT (PKR activating protein)/PRKRA knockout samples were used, along with additional cross-reactive bands. Wild-type and PACT (PKR activating protein)/PRKRA knockout samples were subjected to SDS-PAGE. ab31967 and ab18058 (loading control to Vinculin) were diluted at 1 μg/ml and 1/10000 respectively and incubated overnight at 4°C. 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 1/10000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-PACT (PKR activating protein) / PRKRA antibody (ab31967)

Predicted band size: 34 kDa

false

• WB

Unknown

Western blot - Anti-PACT (PKR activating protein) / PRKRA antibody (AB31967)

Lanes 1-4 : Merged signal (red and green). Green - ab31967 observed at 36 kDa. Red - loading control ab8245 observed at 36 kDa.

ab31967 Anti-PACT (PKR activating protein) / PRKRA antibody was shown to specifically react with PACT in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266806 (knockout cell lysate ab258141) was used. Wild-type and PACT knockout samples were subjected to SDS-PAGE. ab31967 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-PACT (PKR activating protein) / PRKRA antibody (ab31967) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

PRKRA knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human PRKRA (PACT) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-prkra-pact-knockout-hek-293t-cell-line-ab266806'>ab266806</a>)

Lane 3:

K-562 cell lysate at 20 µg

Lane 4:

HepG2 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: 34 kDa

Observed band size: 36 kDa

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