Thermo Scientific
Rabbit anti-MDA5 antibody
catalog # PA5-20337
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Protein extract from cells was prepared in radioimmunoprecipitation assay lysis buffer (sc-24948; Santa Cruz Biotechnology) supplied with PMSF, protease inhibitor mixture, sodium orthovanadate, or RNase inhibitor. Lysates were quantified using a Bio-Rad protein assay kit. Forty micrograms of proteins were subjected to SDS-PAGE and transferred onto nitrocellulose membranes and blocked with 5% non-fat milk in TBS-T (20 mMTris, 500 mMNaCl, and 0.1% Tween 20) at room temperature for 1 h with rocking. The membranes were probed with primary Antibody (1/1000 dilution) overnight at 4°C. After washing with TBS-T, the membranes were incubated with HRP-conjugated secondary Antibodies (Thermo Scientific, Rockford, IL) in 5% non-fat milk/TBS-T at room temperature for 1 h. The protein–Antibody complex was detected by ECL substrate (cat # 32106; Thermo Scientific).
Western Blot
HEK293 cells
4 degrees Celsius
5% milk
Goat anti-rabbit secondary antibody
Room temperature 1 hour
ECL
Knockdown of MDA5 in HEK293 cells
See Supplementary Figure 1B at http://www.jimmunol.org/content/suppl/2014/08/28/jimmunol.1401136.DCSupplemental/JI_1401136_Supplemental_Figures_1.pdf
Works well
NA
Works as expected