Anti-MDA5 antibody [EPR28350-21]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MDA5 antibody [EPR28350-21] (AB315242)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling MDA5 with ab315242 at 1/100 (5.09 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing increased cytoplasmic staining in RAW 264.7 cells treated with IFNβ (2000 U/mL) for 24 hr. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MDA5 antibody [EPR28350-21] (AB315242)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 2000U/ml IFN beta for 24h (Red) / Untreated RAW 264.7 (Green) cells labelling MDA5 with ab315242 at 1/500 dilution (0.1 ug, Red and Green) 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.

• IP

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Immunoprecipitation - Anti-MDA5 antibody [EPR28350-21] (AB315242)

MDA5 was immunoprecipitated from 0.35 mg Mouse spleen whole cell lysate with ab315242 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab315242 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Mouse spleen whole cell lysate

Lane 2 : ab315242 IP in Mouse spleen whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab315242 in mouse spleen whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-MDA5 antibody [EPR28350-21] (ab315242) at 1/30 dilution

All lanes:

Mouse spleen 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: 180s

• WB

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Western blot - Anti-MDA5 antibody [EPR28350-21] (AB315242)

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

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

All lanes:

Western blot - Anti-MDA5 antibody [EPR28350-21] (ab315242) 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 2000U/ml IFN beta for 24 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 1/100000 dilution

Observed band size: 140 kDa,36 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-MDA5 antibody [EPR28350-21] (AB315242)

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

Low expression : testis (PMID : 12015121)

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

The identity of the higher MW band at approximately 250 kDa is unknown.

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

All lanes:

Western blot - Anti-MDA5 antibody [EPR28350-21] (ab315242) at 1/1000 dilution

Lane 1:

Mouse liver tissue lysate at 20 µg

Lane 2:

Mouse testis tissue 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 1/100000 dilution

Observed band size: 140 kDa,90 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-MDA5 antibody [EPR28350-21] (AB315242)

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

Low expression : brain (PMID : 12015121)

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 17267501, 24390337).

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

All lanes:

Western blot - Anti-MDA5 antibody [EPR28350-21] (ab315242) at 1/1000 dilution

Lane 1:

Mouse spleen tissue lysate at 40 µg

Lane 2:

Mouse colon tissue lysate at 40 µg

Lane 3:

Mouse brain tissue lysate at 40 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 1/100000 dilution

Observed band size: 140 kDa,90 kDa,36 kDa

false

Exposure time: 180s