Anti-beta Arrestin 1 antibody [E274]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-beta Arrestin 1 antibody [E274] (AB32099)

Overlay histogram showing PC3 cells stained with unpurified ab32099 (red line). The cells were fixed with 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 (unpurified ab32099, 1/10 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 monoclonal IgG (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in PC3 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-beta Arrestin 1 antibody [E274] (AB32099)

Immunocytochemistry/Immunofluorescence analysis of PC-3 cells labelling beta Arrestin 1 with purified ab32099 at a dilution of 1/150. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1 : primary antibody (1/150) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

• ICC/IF

AbReview30512****

Immunocytochemistry/ Immunofluorescence - Anti-beta Arrestin 1 antibody [E274] (AB32099)

Unpurified ab32099 staining beta Arrestin 1 in C4-2B (Human prostate cancer cell line) by ICC/IF (Immunocytochemistry/immunofluorescence).
Cells were fixed with paraformaldehyde and blocked with 1% serum for 1 hour at 21°C. Samples were incubated with primary antibody (1/100 in diluent) for 1 hour at 21°C. An Alexa Fluor^® 488-conjugated goat anti-rabbit polyclonal IgG (1/200) was used as the secondary antibody.

This image is courtesy of an anonymous Abreview.

• WB

Lab

Western blot - Anti-beta Arrestin 1 antibody [E274] (AB32099)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-beta Arrestin 1 antibody [E274] (ab32099) at 1/1000 dilution

All lanes:

Jurkat 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: 47 kDa

Observed band size: 50 kDa

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• WB

Lab

Western blot - Anti-beta Arrestin 1 antibody [E274] (AB32099)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-beta Arrestin 1 antibody [E274] (ab32099) at 1/2000 dilution

Lane 1:

HEK293 whole cell lysate at 10 µg

Lane 2:

SH-SY5Y whole cell lysate at 10 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: 47 kDa

Observed band size: 50 kDa

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• WB

Lab

Western blot - Anti-beta Arrestin 1 antibody [E274] (AB32099)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-beta Arrestin 1 antibody [E274] (ab32099) at 1/1000 dilution

Lane 1:

C2C12 whole cell lysate at 20 µg

Lane 2:

PC-12 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: 47 kDa

Observed band size: 50 kDa

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• WB

Lab

Western blot - Anti-beta Arrestin 1 antibody [E274] (AB32099)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and Arrb1-KO homozygous mice (Strain ID : T014150).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-beta Arrestin 1 antibody [E274] (ab32099) at 1/1000 dilution

Lane 1:

Wild-type mouse cerebral cortex tissue lysate (male case1) at 20 µg

Lane 2:

Wild-type mouse cerebral cortex tissue lysate (male case2) at 20 µg

Lane 3:

Arrb1 knockout mouse cerebral cortex tissue lysate (male case1) at 20 µg

Lane 4:

Arrb1 knockout mouse cerebral cortex tissue lysate (male case2) at 20 µg

Lane 5:

Wild-type mouse lymph node tissue lysate (male case1) at 20 µg

Lane 6:

Wild-type mouse lymph node tissue lysate (male case2) at 20 µg

Lane 7:

Arrb1 knockout mouse lymph node tissue lysate (male case1) at 20 µg

Lane 8:

Arrb1 knockout mouse lymph node tissue lysate (male case2) 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

Observed band size: 50 kDa,36 kDa

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Exposure time: 180s

• WB

CiteAb

Western blot - Anti-beta Arrestin 1 antibody [E274] (AB32099)

beta Arrestin 1 western blot using anti-beta Arrestin 1 antibody [E274] ab32099. Publication image and figure legend from Wong, A. M., Abrams, M. C., et al., 2015, PLoS One, PubMed 25803606.

ab32099 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 ab32099 please see the product overview.

Estradiol (E2)-induced ERK1/2 phosphorylation in N-38 cells was attenuated in cells transfected with Arrb1 siRNA.(A) Western blots from scrambled siRNA-transfected N-38 cells probed with Arrb1 and GAPDH. (B) Arrb1 western blots from scrambled siRNA-transfected N-38 cells showing knockdown of Arrb1 protein. (C) Representative western blots of levels of ERK1/2 (p44/42 MAPK) phosphorylation in scrambled siRNA-transfected N-38 cells and (D) N-38 cells transfected with Arrb1 siRNA treated with estradiol (E2) for 0–120 min. (E) Densitometric analysis of the optical density from western blots of phosphorylated ERK1/2 (p44/42) protein bands normalized to the optical densities of total ERK1/2 protein in scrambled vs. Arrb1 siRNA-transfected cells. Controls for each group (cells not treated with E2) was set at 100%, and E2 treatments were expressed as a percentage of the control. Values represent means (n = 5) ± SEM. * (p<0.05). **(p<0.005).

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