Anti-Pyrin 抗体 [EPR18676]

• IP

Supplier Data

Immunoprecipitation - Anti-Pyrin antibody [EPR18676] (AB195975)

Pyrin was immunoprecipitated from DC2.4 (Human immature dendritic cell line) stable Pyrin expression whole cell lysate with ab195975 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab195975 at 1/1000 dilution. Goat anti-Rabbit IgG (H+L), was used as secondary antibody at 1/5000 dilution.

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

Exposure time : 30 seconds.

The image was kindly provided by Dr Feng Shao's lab, NIBS.

All lanes:

Immunoprecipitation - Anti-Pyrin antibody [EPR18676] (ab195975) at 1/100 dilution

Lane 1:

DC2.4 whole cell lysate

Lanes 2 - 3:

DC2.4 stable Pyrin expression whole cell lysate

Lane 4:

ab195975 IP in DC2.4 whole cell lysate

Lane 5:

ab195975 IP in DC2.4 stable Pyrin expression whole cell lysate

Lane 6:

Mock IP (without ab195975) in DC2.4 stable Pyrin expression whole cell lysate

Predicted band size: 86 kDa

false

Exposure time: 30s

• IP

Lab

Immunoprecipitation - Anti-Pyrin antibody [EPR18676] (AB195975)

Pyrin was immunoprecipitated from mouse spleen tissue whole cell lysate 10 μg with ab195975. Western blot was performed on the immunoprecipitate using ab195975 at 1/1000 dilution. Capture antibody, 1/30 dilution (2μg in 0.35mg lysates).

Rabbit monoclonal IgG (ab172730) Isotype Control was used instead of ab195975 in mouse spleen tissue whole cell lysate (Lane 3).

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

All lanes:

Immunoprecipitation - Anti-Pyrin antibody [EPR18676] (ab195975) at 1/1000 dilution

Lanes 1 - 2:

Mouse spleen tissue whole cell lysate at 25 µg

Lane 3:

Rabbit monoclonal IgG (<a href='/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab195975 in mouse spleen tissue whole cell lysate

Secondary

All lanes:

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

Observed band size: 86-110 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-Pyrin antibody [EPR18676] (AB195975)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200,000 dilution.

Negative control : heart.

To minimize protein degradation, cells/tissues were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

The identity of the band between 75 kDa and 50 kDa is unknown.

All lanes:

Western blot - Anti-Pyrin antibody [EPR18676] (ab195975) at 1/1000 dilution

Lane 1:

Mouse spleen tissue lysate at 20 µg

Lane 2:

Mouse heart tissue lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 86-110 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-Pyrin antibody [EPR18676] (AB195975)

Blocking/Dilution buffer : 5% milk/TBST.

The image was kindly provided by our collaborator Dr Feng Shao's lab, NIBS.

All lanes:

Western blot - Anti-Pyrin antibody [EPR18676] (ab195975) at 1/1000 dilution

Lane 1:

Bone marrow derived-macrophage of Pyrin -/- mice at 5 µg

Lane 2:

Bone marrow derived-macrophage of Pyrin -/- mice stimulated with LPS at 5 µg

Lane 3:

Bone marrow derived-macrophage of Pyrin -/- mice stimulated with TNF alpha at 5 µg

Lane 4:

Bone marrow derived-macrophage of wild type C57/B6 mice at 5 µg

Lane 5:

Bone marrow derived-macrophage of wild type C57/B6 mice stimulated with LPS at 5 µg

Lane 6:

Bone marrow derived-macrophage of wild type C57/B6 mice stimulated with TNF alpha at 5 µg

Secondary

All lanes:

Goat anti-Rabbit IgG (H+L) at 1/5000 dilution

Predicted band size: 86 kDa

Observed band size: 110 kDa

false

Exposure time: 60s

• WB

Supplier Data

Western blot - Anti-Pyrin antibody [EPR18676] (AB195975)

Blocking/Dilution buffer : 5% NFDM/TBST.

The lysates were kindly provided by our collaborator Dr Feng Shao's lab, NIBS.

All lanes:

Western blot - Anti-Pyrin antibody [EPR18676] (ab195975) at 1/50 dilution

Lane 1:

Bone marrow derived-macrophage Pyrin -/- mice at 10 µg

Lane 2:

Bone marrow derived-macrophage Pyrin -/- mice stimulated with LPS at 10 µg

Lane 3:

Bone marrow derived-macrophage of wild type C57/B6 mice at 10 µg

Lane 4:

Bone marrow derived-macrophage of wild type C57/B6 mice stimulated with LPS at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/2000 dilution

Predicted band size: 86 kDa

false

Exposure time: 30s