Anti-CD80 antibody [EPR1157(2)]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD80 antibody [EPR1157(2)] (AB134120)

Tissue Microarrays stained for "Anti-CD80 antibody [EPR1157(2)]" using "ab134120"in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes. The sections were incubated with ab134120 for 30 mins at room temperature followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CD80 antibody [EPR1157(2)] (AB134120)

Flow cytometry overlay histogram showing wild-type Raji (green line) and CD80 knockout Raji stained with ab134120 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10μg/ml human IgG and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab134120) (1x 106 in 100μl at 0.2 μg/ml (1/10150)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (wild-type Raji - black line, CD80 knockout Raji - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in Raji Fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• IP

Lab

Immunoprecipitation - Anti-CD80 antibody [EPR1157(2)] (AB134120)

CD80 was immunoprecipitated from 0.35 mg Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate with ab134120 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab134120 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP) (ab131366) was used at 1/5000 dilution.

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

Lane 1:

Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate 10 μg

Lane 2:

Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab134120 in Raji whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>)

Observed band size: 60 kDa

false

Exposure time: 6s

• WB

Lab

Western blot - Anti-CD80 antibody [EPR1157(2)] (AB134120)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-CD80 antibody [EPR1157(2)] (ab134120) at 1/2500 dilution

All lanes:

Raji cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 33 kDa

Observed band size: 60 kDa

false

• WB

Unknown

Western blot - Anti-CD80 antibody [EPR1157(2)] (AB134120)

All lanes:

Western blot - Anti-CD80 antibody [EPR1157(2)] (ab134120) at 1/1000 dilution

Lane 1:

Raji cell lysate at 10 µg

Lane 2:

Ramos cell lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP conjugated antibody at 1/2000 dilution

Predicted band size: 33 kDa,43 kDa

Observed band size: 43 kDa,60 kDa

false

• WB

Lab

Western blot - Anti-CD80 antibody [EPR1157(2)] (AB134120)

Lanes 1 - 3 : Merged signal (red and green). Green - ab134120 observed at 55 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab134120 was shown to react with CD80 in wild-type Raji cells in western blot with loss of signal observed in CD80 knockout sample. Wild-type and CD80 knockout Raji cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab134120 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CD80 antibody [EPR1157(2)] (ab134120) at 1/1000 dilution

Lane 1:

Wild-type Raji (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Lane 2:

CD80 knockout Raji (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human CD80 knockout Raji cell line (<a href='/en-us/products/cell-lines/human-cd80-knockout-raji-cell-line-ab273873'>ab273873</a>)

Lane 3:

Ramos (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Predicted band size: 33 kDa

Observed band size: 55 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD80 antibody [EPR1157(2)] (AB134120)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling CD80 with ab134120 at a dilution of 1/1000. Heat mediated antigen retrieval was performed using Trs/EDTA buffer pH9 (ab93684). ImmunoHistoProbe one step HRP Polymer was used. Counter stained with hematoxylin.

The image shows cytoplasmic and membrane staining.