MBP | Maltose Binding Protein (monoclonal)

Insect cell lysate was prepared in buffer: HEPES 20mM, NaCl 150 mM, LMNG-CHS, amylase resin was used for purification, elution was conducted with the same buffer with maltose. 20 µg/well of eluted protein or fresh lysate were denatured at 95°C for 5 min and separated on 10% SDS-PAGE and blotted 1h to nitrocellulose (pore size of 0.45 um), using wet transfer. Blot was blocked with 3% milk for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 in TBS-T ON/4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:1 000 in for 1h/RT with agitation. The blot was washed as above and developed for 5 min with AgriseraECLSuperBright. Exposure time was 20-30 seconds.

Courtesy of Natalia Piłka, Laboratory of Lipid Biochemistry, Polish Academy of Sciences, Warsaw, Poland

MBP (Maltose binding protein) is encoded by the malE gene of E.coli and is a commonly used tag when studying protein expression using a wide range of applications. MBP tag enables easy purification of proteins from bacterial extracts under mild conditions.