Anti-Trx Tag | Thioredoxin 1 Fusion protein

Analysis of production of protein of interest (POI): H10-TRX-POIoverexpressed in E. Coli BL21 (DE3) Rosetta 

Samples:
1- 10 µl of total fraction of cell lysate, before induction.
2- 10 µl of total fraction of cell lysate of IPTG (0.05mM) induced (ON, 16ºC) BL21 (DE3) Rosetta.
3- 10 µl of soluble fraction of cell lysate of IPTG (0.05mM) induced (ON, 16ºC) BL21 (DE3) Rosetta.
4- 10 µl of insoluble pellet fraction of cell lysate of IPTG (0.05mM) induced (ON, 16ºC) BL21 (DE3) Rosetta.
5- 10 µl of total fraction of cell lysate of IPTG (0.1mM) induced (ON, 16ºC) BL21 (DE3) Rosetta.
6- 10 µl of soluble fraction of cell lysate of IPTG (0.1mM) induced (ON, 16ºC) BL21 (DE3) Rosetta.
7- 10 µl of insoluble pellet fraction of cell lysate of IPTG (0.1mM) induced (ON, 16ºC) BL21 (DE3) Rosetta.
8- 10 µl of soluble fraction of negative control H10-POI

10 µl of protein extract from BL21 (DE3) Rosetta (all bacteria pellets were normalized to final DO of 6.5).  Exact buffer components were: Tris-HCl 50 mM, NaCl 500mM, 10% glycerol, pH:8 and denatured with 2X buffer (Tris-HCl 125 mM, 20% glycerol, SDS 4%, BeOH, 2% and blue bromophenol 0.001%) at 95°C 5min.  Samples were separated in the cold on 10 % SDS-PAGE and blotted for 1h 30min nitrocellulose (0.45 µm), using: wet transfer in the cold. Blot was blocked with 5 % milk for: 2h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 2000 for ON/4°C with agitation. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed 3 times for 10 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:10 000 in 5% nonfat milk TBT-T for 1 h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent. Exposure time was 30 seconds.

Dr.Pablo Cerdán, Fundación Instituto Leloir, Argentina