Hrp conjugated goat anti rabbit secondary antibody

BMDM protein lysates were quantified by BCA analysis (Thermo Fisher Scientific, cat # 23225), diluted to a concentration of 1 mg/mL, and mixed 1:1 with 2x Laemmli Buffer (Bio-Rad Laboratories, cat # 161-0737). 10 µg of protein was loaded from each sample into separate wells of a 4–15% polyacrylamide gel (Bio-Rad Laboratories, cat # 456-8084). Electrophoresis was performed in 1x Tris/Glycine Buffer (Bio-Rad Laboratories, cat # 1610734) at 100 V, and protein was transferred to a PVDF membrane (Bio-Rad Laboratories, cat # 1620177) overnight at 35 V and 4 °C. Following the transfer, non-specific protein binding was blocked by incubation of the membrane in 5% non-fat milk for 30 min. The membrane was then incubated with the primary anti-LDHA antibody (ProteinTech, cat # 19987-1-AP) diluted 1:1000 in 5% non-fat milk at RT for one hour. The membrane was washed three times in TBST and incubated with the HRP-conjugated goat-anti-rabbit secondary antibody (Kindle Biosciences, cat # R1004) diluted 1:1000 in 5% non-fat milk at RT for one hour. The membrane was then covered in KwikKwant HRP Substrate Solutions A + B (Kindle Biosciences, R1004) and images on a KwikKwant Imager (Kindle Biosciences). Images were quantified with the Fiji/ImageJ^{65 (link)} gel analysis tool, version 1.52p.