Anti-B MyB (phospho T487) antibody [EPR2204Y]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-B MyB (phospho T487) antibody [EPR2204Y] (AB76009)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling B MyB (phospho T487) with primary antibody anti-B MyB (phospho T487) (ab76009) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150081) secondary antibody at 1/1000 dilution. Confocal image showing nuclear staining in HeLa cells and no staining in HeLa cells with Alkaline Phosphatase treatment 37℃ for 1 hour. Anti-alpha Tubulin antibody (DM1A) - Microtubule Marker (Alexa Fluor^® 594) (ab195889) was used to counterstain tubulin at 1/200 dilution. The nuclear counter stain is DAPI (blue).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-B MyB (phospho T487) antibody [EPR2204Y] (AB76009)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells treated with Alkaline Phosphatase overnight (Left) and untreated HeLa cells (Right) labeling B MyB (phospho T487) with ab76009 at 1/1000 dilution (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150081) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-B MyB (phospho T487) antibody [EPR2204Y] (AB76009)

Immunohistochemistry analysis of paraffin-embedded Human colon cancer tissue sections labelling B MyB (phospho T487) with ab76009 at 1/8000 dilution. The section was incubated with ab76009 for 30 mins at room temperature. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Sections were counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.
Nuclear staining on Human colon cancer tissue without alkaline phosphatase treatment (Image A); No signal was detected when tissues were treated with alkaline phosphatase (Image B). The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-B MyB (phospho T487) antibody [EPR2204Y] (AB76009)

Immunohistochemistry analysis of paraffin-embedded Human lung cancer tissue sections labelling B MyB (phospho T487) with ab76009 at 1/8000 dilution. The section was incubated with ab76009 for 30 mins at room temperature. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Sections were counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.
Nuclear staining on Human lung cancer tissue without alkaline phosphatase treatment (Image A); No signal was detected when tissues were treated with alkaline phosphatase (Image B). The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• WB

Lab

Western blot - Anti-B MyB (phospho T487) antibody [EPR2204Y] (AB76009)

Blocking buffer and concentration : 5% NFDM/TBST Diluting buffer and concentration : 5% NFDM/TBST

Lanes 1 - 2:

Western blot - Anti-B MyB (phospho T487) antibody [EPR2204Y] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/b-myb-phospho-t487-antibody-epr2204y-bsa-and-azide-free-ab232521'>ab232521</a>) at 1/1000 dilution

Lanes 1 - 2:

Western blot - Anti-B MyB (phospho T487) antibody [EPR2204Y] (ab76009) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) nuclear fraction lysate at 15 µg

Lane 2:

HeLa nuclear fraction lysate treated with Alkaline Phosphatase for 1 hour at 15 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: 79 kDa

Observed band size: 100 kDa

false

Exposure time: 120s

• WB

Lab

Western blot - Anti-B MyB (phospho T487) antibody [EPR2204Y] (AB76009)

Blocking buffer and concentration : 5% NFDM/TBST Diluting buffer and concentration : 5% NFDM/TBST

Lanes 1 - 2:

Western blot - Anti-B MyB (phospho T487) antibody [EPR2204Y] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/b-myb-phospho-t487-antibody-epr2204y-bsa-and-azide-free-ab232521'>ab232521</a>) at 1/1000 dilution

Lanes 1 - 2:

Western blot - Anti-B MyB (phospho T487) antibody [EPR2204Y] (ab76009) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) nuclear fraction lysate at 15 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) without nuclear fraction lysate at 15 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: 79 kDa

Observed band size: 100 kDa

false

Exposure time: 60s

• WB

Lab

Western blot - Anti-B MyB (phospho T487) antibody [EPR2204Y] (AB76009)

Blocking buffer and concentration : 5% NFDM/TBST Diluting buffer and concentration : 5% NFDM/TBST

Lanes 1 - 2:

Western blot - Anti-B MyB (phospho T487) antibody [EPR2204Y] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/b-myb-phospho-t487-antibody-epr2204y-bsa-and-azide-free-ab232521'>ab232521</a>) at 1/1000 dilution

Lanes 1 - 2:

Western blot - Anti-B MyB (phospho T487) antibody [EPR2204Y] (ab76009) at 1/1000 dilution

Lane 1:

293T (Human embryonic kidney epithelial cell) transfected with empty vector (vector control), containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 2:

293T transfected with human B MyB (WT) expression vector containing a myc-His-tag®, 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: 79 kDa

Observed band size: 100 kDa

false

Exposure time: 20s

• Dot

Lab

Dot Blot - Anti-B MyB (phospho T487) antibody [EPR2204Y] (AB76009)

Dot blot analysis of B MyB (pT487) phospho peptide (lane 1) and B MyB non-phospho peptide (lane 2) with ab76009 at a 1/1000 dilution. Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) was used as the secondary antibody at a dilution of 1/20,000.
Blocking and dilution buffer : 5% NFDM/TBST
Exposure time : 3 minutes