Anti-PKR antibody [Y117]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-PKR antibody [Y117] (AB32506)

Intracellular Flow Cytometry analysis of MCF-7 (human breast carcinoma) cells labeling PKR with purified ab32506 at 1/20 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-PKR antibody [Y117] (AB32506)

Immunofluorescent staining of HeLa cells using unpurified ab32506 at 1/250 dilution.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PKR antibody [Y117] (AB32506)

ab32506 staining PKR in human liver carcinoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1 : PBS in place of primary antibody.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-PKR antibody [Y117] (AB32506)

ab32506 staining PKR in wild-type HAP1 cells (top panel) and PKR knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% 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 with ab32506 at 1/400 dilution and ab7291 at 1ug/ml concentration overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to Mouse IgG (Alexa Fluor® 594) (ab150117) at 2ug/ml (shown in pseudo-color red). Nuclear DNA was labelled in blue with DAPI.

This product also gave a positive signal under the same testing conditions in HAP1 cells fixed with 4% formaldehyde (10 min).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PKR antibody [Y117] (AB32506)

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using unpurified ab32506 at 1/100 dilution.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-PKR antibody [Y117] (AB32506)

ab32506 staining PKR in MCF-7 (human breast carcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody. ab7291 and ab150120 were used as counterstains for primary antibody ab32506 and secondary antibody ab150077 respectively and DAPI was used as a nuclear counterstain.

Negative control 1 : Rabbit primary antibody and anti-mouse secondary antibody (ab150120)
Negative control 2 : Mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077)

• IP

Unknown

Immunoprecipitation - Anti-PKR antibody [Y117] (AB32506)

ab32506 immunoprecipitating PKR. 10μg of cell lysate was incubated with primary antibody at a dilution of 1/40 and VeriBlot for IP Detection Reagent (HRP) (ab131366) at a dilution of 1/10000.

Lane 1 : HEK293 (human embryonic kidney) whole cell lysate (10ug)
Lane 2 : HEK293 (human embryonic kidney) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab32506 in HEK293 (human embryonic kidney) whole cell lysate

All lanes:

Immunoprecipitation - Anti-PKR antibody [Y117] (ab32506)

Predicted band size: 62 kDa

Observed band size: 40 kDa,68 kDa

false

• WB

Unknown

Western blot - Anti-PKR antibody [Y117] (AB32506)

Lanes 1, 3 and 5 : PKR knockout HAP1 cell lysate (20 μg)
Lanes 2, 4 and 6 : Wild-type HAP1 cell lysate (20 μg)
Lanes 1 and 2 : Green signal from target - ab32506 observed at 62 kDa
Lanes 3 and 4 : Red signal from loading control - ab8245 observed at 37 kDa
Lanes 5 and 6 : Merged (red and green) signal
ab32506 was shown to specifically react with PKR when PKR knockout samples were used. Wild-type and PKR knockout samples were subjected to SDS-PAGE. ab32506 and ab8245 (loading control to GAPDH) were diluted 1/10 000 and 1/2000 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 1/10 000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-PKR antibody [Y117] (ab32506)

Predicted band size: 62 kDa

false

• WB

Lab

Western blot - Anti-PKR antibody [Y117] (AB32506)

Purified format.

All lanes:

Western blot - Anti-PKR antibody [Y117] (ab32506) at 1/20000 dilution

Lane 1:

MCF-7 (human breast carcinoma) whole cell lysates at 20 µg

Lane 2:

HEK293 (human embryonic kidney) whole cell lysates 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 1/100000 dilution

Predicted band size: 62 kDa

Observed band size: 68 kDa

false

• WB

Unknown

Western blot - Anti-PKR antibody [Y117] (AB32506)

All lanes:

Western blot - Anti-PKR antibody [Y117] (ab32506) at 1/10000 dilution

All lanes:

MCF-7 cell lysate

Predicted band size: 62 kDa

Observed band size: 68 kDa

false

• WB

CiteAb

Western blot - Anti-PKR antibody [Y117] (AB32506)

PKR western blot using anti-PKR antibody [Y117] ab32506. Publication image and figure legend from Liu, G. & Zhang, W., 2018, Braz J Med Biol Res, PubMed 29898032.

ab32506 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab32506 please see the product overview.

Effect of HOTAIR on PKR expression; A, qRT-PCRrevealed overexpression of HOTAIR promoted the expression of PKR;B, Western blot analysis revealedoverexpression of HOTAIR promoted the expression of PKR. Data arereported as means±SD. *p<0.05; **p<0.01 (ANOVA). HOTAIR : HOXantisense intergenic RNA; PKR : RNA-dependent protein kinase; qRT-PCR : quantitative real-time polymerase chain reaction.

false