Anti-IL-10 antibody [EPR27280-88]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-IL-10 antibody [EPR27280-88] (AB310329)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Isotype (Left) / RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/ml LPS for 6h and then add 300ng/ml BFA for 3h (middle) / untreated RAW 264.7 (Right) cells labelling IL-10 with ab310329 at 1/500 dilution (0.1 ug)/Right (Red) compared with a Rabbit monoclonal IgG (ab172730) / Left 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.

• IP

Supplier Data

Immunoprecipitation - Anti-IL-10 antibody [EPR27280-88] (AB310329)

IL-10 was immunoprecipitated from 0.35 mg RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100 ng/mL LPS for 6 hours, 300ng/mL Brefeldin A was then added for additional 3 hours whole cell lysate with ab310329 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab310329 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100 ng/mL LPS for 6 hours, 300ng/mL Brefeldin A was then added for additional 3 hours whole cell lysate Lane 2 : ab310329 IP in RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100 ng/mL LPS for 6 hours, 300ng/mL Brefeldin A was then added for additional 3 hours whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab310329 in RAW264.7 treated with 100 ng/mL LPS for 6 hours, 300ng/mL Brefeldin A was then added for additional 3 hours whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-IL-10 antibody [EPR27280-88] (ab310329) at 1/30 dilution

All lanes:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100 ng/mL LPS for 6 hours, 300ng/mL Brefeldin A was then added for additional 3 hours 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>) at 1/5000 dilution

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-IL-10 antibody [EPR27280-88] (AB310329)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. The weaker band observed at 15kDa is the deglycosylated form of IL-10. In western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

All lanes:

Western blot - Anti-IL-10 antibody [EPR27280-88] (ab310329) at 1/1000 dilution

Lane 1:

Untreated RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with /mL LPS for 6 hours, 300ng/mL Brefeldin A was then added for additional 3 hours whole cell lysate at 20 µg

Lane 2:

RAW264.7 treated with /mL LPS for 6 hours, 300ng/mL Brefeldin A was then added for additional 3 hours whole cell lysate treated with N-glycosidase F at 20 µg

Secondary

All lanes:

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

Observed band size: 18 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-IL-10 antibody [EPR27280-88] (AB310329)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. The expression of IL-10 is upregulated in response to LPS in treated RAW 264.7 (PMID : 23012430, 15218058) but not in non-differentiated NIH/3T3 cells. In western blot, anti-IL-6 antibody (ab290735) staining at 1/1000 dilution. Anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

All lanes:

Western blot - Anti-IL-10 antibody [EPR27280-88] (ab310329) at 1/1000 dilution

Lane 1:

Untreated RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

RAW264.7 treated with 100 ng/mL LPS for 6 hours, 300ng/mL Brefeldin A was then added for additional 3 hours whole cell lysate at 20 µg

Lane 3:

Untreated NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 4:

NIH/3T3 treated with 100 ng/mL LPS for 6 hours, 300ng/mL Brefeldin A was then added for additional 3 hours whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 20 kDa

Observed band size: 18 kDa

false

Exposure time: 3min