SDS Page-Gel for all the bovine casein inserts

17 January 2015: SDS Page-Gel All 5 Bovine Casein Inserts

Location: BioCurious

Participants: Lafia, Johan, Meenakshi, Emi, Rachel

Notes: Emi

Aims: Confirmation of bovine casein production from liquid culture supernatant of transformed yeast cells

Materials

Gel loading tips (BioRad)

Tris-Glycine SDS Running Buffer 10X stock (Novex Life Tech, Catalog number: LC2675)

Tris-Glycine Mini Gels 4-20% 1.0mm x 10 wells/gel, (Catalog number: EC6025Box, Lot 14082110, received September 2014, possibly stored at -20C for a period of time)

Protein ladder (10-250 kDa) (NEB, Catalog number: p7703S)

Micellular casein powder from CCL stock

Chymosin solution (CHY-MAX, CCL stock)

Gel box and power supply from BioCurious

Bov alpha s1 (1) Transformed liqid culture

Bov beta B (8) Transformed liquid culture

Bov alpha S2 (3) Transformed liquid culture

Bov beta A2 (6B) Transformed liquid culture

Bov kappa (7F) Transformed liquid culture

Transformed negative control

Chymosin + casein (Chymosin cuts casein and separates it to 2 bands)

Protocol

Centrifuge the falcon tubes, 5 minutes 2800xg

Take 500ul of distilled water in eppendorf tube + 1 tiny drop of casein (use 200ul hp take it) - 2times

Take 15ul of chymosin +1 tiny drop of casein in 250ul of water

Take 250ul of 7F (Bov Kappa) + 15ul of Chymosinin Take 250ul of water and 15ul of chymosin

Take 40ul of 1, 8, 3B, 6B, +7F also the negative control to eppendorf tubes

Preparation fo 20x running buffer

Pour 50ul of 10x running buffer; make it to 450ul by adding the distilled water - total 500ul

20ul of loading dye to each tubes except casein; chymosin and casein; chymosin and 7F

Thus we have to add total 120ul of dye

Boil all the tubes for 10 mins at 95C (in order to denature the protein structure to linear)

Results

Gel#2