Anti-B7-H6 antibody [EPR23470-210] - BSA and Azide free

• IP

Unknown

Immunoprecipitation - Anti-B7-H6 antibody [EPR23470-210] - BSA and Azide free (AB275029)

This data was developed using ab253180, the same antibody clone in a different buffer formulation.

B7-H6 was immunoprecipitated from 0.35 mg HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate with ab253180 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab253180 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate 10 μg

Lane 2 : ab253180 IP in HepG2 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab253180 in HepG2 whole cell lysate

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

Exposure time : 41 seconds.

The lower bands are likely to be the deglycosylated form of B7-H6

All lanes:

Immunoprecipitation - Anti-B7-H6 antibody [EPR23470-210] (<a href='/en-us/products/primary-antibodies/b7-h6-antibody-epr23470-210-ab253180'>ab253180</a>)

Predicted band size: 51 kDa

Observed band size: 80 kDa

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• WB

Lab

Western blot - Anti-B7-H6 antibody [EPR23470-210] - BSA and Azide free (AB275029)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab253180).

Western blot : Anti-B7-H6 antibody [EPR23470-210] ab253180 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 70-80 kDa in Wild-type Raji UNBOILED cell lysates with no signal observed at this size in NCR3LG1 knockout Raji UNBOILED cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc Milk in TBS-0.1 % Tween^™ 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-B7-H6 antibody [EPR23470-210] (<a href='/en-us/products/primary-antibodies/b7-h6-antibody-epr23470-210-ab253180'>ab253180</a>) at 1/1000 dilution

Lane 1:

Wild-type Raji UNBOILED whole cell lysate at 20 µg

Lane 2:

Western blot - Human NCR3LG1 knockout Raji cell line (<a href='/en-us/products/cell-lines/human-ncr3lg1-knockout-raji-cell-line-ab290414'>ab290414</a>) at 20 µg

Lane 3:

HepG2 UNBOILED whole cell lysate at 20 µg

Lane 4:

K562 UNBOILED whole cell lysate at 20 µg

Lane 5:

Daudi UNBOILED whole cell lysate at 20 µg

Lane 6:

SK-OV-3 UNBOILED whole cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 51 kDa

Observed band size: 70-80 kDa

false

• WB

Unknown

Western blot - Anti-B7-H6 antibody [EPR23470-210] - BSA and Azide free (AB275029)

This data was developed using ab253180, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.

Negative control : MCF7.

The expression profile is consistent with the literature (PMID : 22851709).
B7-H6 is a glycoprotein and can be de-glycosylated by PNGase F. The molecular mass observed is consistent with the literature (PMID : 24780758).

All lanes:

Western blot - Anti-B7-H6 antibody [EPR23470-210] (<a href='/en-us/products/primary-antibodies/b7-h6-antibody-epr23470-210-ab253180'>ab253180</a>) at 1/1000 dilution

Lane 1:

Untreated HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

HepG2 whole cell lysate treated with PNGase F 20 at 20 µg

Lane 3:

K562 (human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate at 20 µg

Lane 4:

MCF7 (human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 51 kDa

Observed band size: 50 kDa,80 kDa

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