Anti-CD80 antibody [RM1104]

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-CD80 antibody [RM1104] (AB315857)

Flow cytometric analysis of CD80 KO Raji (human CD80 knockout Burkitt's lymphoma B lymphocyte, Left) / Parental Raji (Right) cells labelling CD80 with ab315857 at 1/5000 dilution (0.01 ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

In flow cytometry, ab315857 (upper) shows positive staining on Raji and no staining on CD80 KO Raji, while ab254579 (lower) was shown non-specific binding CD80.

The upper images are applied with Anti-CD80 antibody (ab315857) at 1/5000 dilution and the lower images are applied with Anti-CD80 antibody (ab254579) at 1/50 dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-CD80 antibody [RM1104] (AB315857)

CD80 was immunoprecipitated from 0.35 mg Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate with ab315857 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab315857 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

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

All lanes:

Immunoprecipitation - Anti-CD80 antibody [RM1104] (ab315857) at 1/1000 dilution

Lane 1:

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

Lane 2:

ab315857 at 1/30 IP in Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/ko/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab315857 in Raji whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/ko/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

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Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-CD80 antibody [RM1104] (AB315857)

Western blot : Anti-CD80 antibody (panel A : ab315857, panel B : ab254579) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5](ab8245) loading control staining at 1/20000 dilution, shown in red.

In Western blot, ab315857 (panel A) was shown to bind specifically to CD80, while ab254579 (panel B) was shown non-specific binding CD80. Target of interest was observed at 55-60 kDa in wild-type Raji cell lysates (lane 1) with no signal observed at this size in CD80 knockout cell line (lane 2, knockout cell line ab273873/knockout cell lysate ab273827). To generate this image, samples were first run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in a fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed in TBS-T, incubated with secondary antibodies Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution for 1 h at room temperature, washed again then imaged.

All lanes:

Western blot - Anti-CD80 antibody [RM1104] (ab315857) at 1/1000 dilution

Lane 1:

Wild-type Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 2:

CD80 knockout Raji whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (800CW) and Goat Anti-Mouse IgG H&L (680RD) at 1/20000 dilution

Observed band size: 55-60 kDa

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• WB

Supplier Data

Western blot - Anti-CD80 antibody [RM1104] (AB315857)

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

Negative control : HUVEC (PMID : 15064090), 293T.

The molecular mass observed is consistent with what has been described in the literature (PMID : 7544014, PMID : 9079798); 55-60 kDa is due to glycosylation (PMID : 9654121). The band over 200 kDa is unknown band.

This blot was developed using a high sensitivity ECL substrate (Lanes 6-7). The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

Exposure time : Lane 1-5 : 70 seconds Lane 6-7 : 59 seconds

All lanes:

Western blot - Anti-CD80 antibody [RM1104] (ab315857) at 1/1000 dilution

Lane 1:

Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 2:

Daudi (human Burkitts lymphoma lymphoblast) whole cell lysate at 20 µg

Lane 3:

HDLM-2 (human Hodgkin lymphoma cell) whole cell lysate at 20 µg

Lane 4:

HUVEC (human umbilical vein endothelial cell) whole cell lysate at 20 µg

Lane 5:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 6:

Human tonsil tissue lysate at 20 µg

Lane 7:

Human spleen tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 55-60 kDa,36 kDa

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• WB

Lab

Western blot - Anti-CD80 antibody [RM1104] (AB315857)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. Negative control : P815 (PMID : 9237108). The band around 100 kDa is unknown band. In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control  (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-CD80 antibody [RM1104] (ab315857) at 1/1000 dilution

Lane 1:

C2C12 (mouse myoblast) whole cell lysate at 20 µg

Lane 2:

P815 (mouse mastocytoma mast Cell) whole cell lysate at 20 µg

Observed band size: 55-60 kDa

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Exposure time: 70s