Anti-PLCG 2 antibody [EPR5914-34]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-PLCG 2 antibody [EPR5914-34] (AB133522)

Flow Cytometry analysis of Human Peripheral Blood Mononuclear Cell (PBMC) cells labeling PLCG 2 with purified ab133522 at 1/1000 dilution (1 μg/mL) (Red). Cells were fixed with 2% Paraformaldehyde and permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150081) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Left).
Cells were surface stained with anti-CD19 conjugated to PE/Cy7, then fixed with 2% PFA followed by intracellularly stained with rabbit IgG or ab133522. Gated on lymphocytes.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PLCG 2 antibody [EPR5914-34] (AB133522)

Immunohistochemistry analysis of Paraffin Embedded Human gastric adenocarcinoma tissue labelling PLCG2 with ab133522 at 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• IP

Lab

Immunoprecipitation - Anti-PLCG 2 antibody [EPR5914-34] (AB133522)

This data was developed using ab248535, the same antibody clone in a different buffer formulation. Western blot : antibody description (ab248535) staining at 10,000 dilution, shown in Black. In Western blot, ab248535 was shown to bind specifically to PLCG2. An enriched band was observed at 160 kDa in samples pulled down with ab248535 with no band observed in Isotype control sample. Following IP, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 before development with a high-sensitivity ECL substrate kit and imaged. Secondary antibodies used was VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution.

Lanes 1 - 2:

Immunoprecipitation - Anti-PLCG 2 antibody [EPR5914-34] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/plcg-2-antibody-epr5914-34-bsa-and-azide-free-ab248535'>ab248535</a>) at 1/10000 dilution

Lanes 1 - 2:

Immunoprecipitation - Anti-PLCG 2 antibody [EPR5914-34] (ab133522)

Lane 1:

HAP1 Input cell lysate at 10 µg

Lane 2:

HAP1 pulldown with <a href='/en-us/products/primary-antibodies/plcg-2-antibody-epr5914-34-bsa-and-azide-free-ab248535'>ab248535</a> at 10 µg

Lane 3:

HAP1 pulldown with <a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a> Isotype control at 10 µg

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>) at 1/1000 dilution

Observed band size: 160 kDa

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Exposure time: 4min

• WB

Lab

Western blot - Anti-PLCG 2 antibody [EPR5914-34] (AB133522)

Anti-PLCG2 antibody [EPR5914-34] (ab133522) staining at 1/10000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab133522 was shown to bind specifically to PLCG2. A band was observed at 148 kDa in wild-type HAP1 cell lysates with no signal observed at this size in PLCG2 knockout cell line. To generate this image, wild-type and PLCG2 knockout HAP1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-PLCG 2 antibody [EPR5914-34] (ab133522) at 1/10000 dilution

All lanes:

Western blot 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

Predicted band size: 148 kDa

Observed band size: 148 kDa

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• WB

Lab

Western blot - Anti-PLCG 2 antibody [EPR5914-34] (AB133522)

Western blot : Anti-PLCG2 antibody [EPR5914-34] (ab133522) staining at 1/10000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab133522 was shown to bind specifically to PLCG2. A band was observed at 150 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in PLCG2 knockout cell line. To generate this image, wild-type and PLCG2 knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-PLCG 2 antibody [EPR5914-34] (ab133522) at 1/10000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

Western blot - Human PLCG2 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-plcg2-knockout-hct116-cell-line-ab301166'>ab301166</a>)

Lane 2:

PLCG2 knockout HCT 116 cell lysate at 20 µg

Secondary

Lanes 1 - 2:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 2:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 150 kDa

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• WB

Lab

Western blot - Anti-PLCG 2 antibody [EPR5914-34] (AB133522)

All lanes:

Western blot - Anti-PLCG 2 antibody [EPR5914-34] (ab133522) at 1/10000 dilution

Lane 1:

Raji cell lysates at 10 µg

Lane 2:

Ramos cell lysates at 10 µg

Secondary

All lanes:

HRP-labelled Goat anti-Rabbit at 1/2000 dilution

Predicted band size: 148 kDa

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• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-PLCG 2 antibody [EPR5914-34] (AB133522)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.