Western Blot of bovine casein

May 2, 2015: Western Blot of Bovine Casein

Location: BioCurious

Participants: Priyanka, Johan, Advait, Maria, Patrik

Notes: Priyanka, Johan, Advait

Aims: Confirm presence of bovine casein protein

Material: for SDS page:

SDS (10%) catalog number: SDS10-100

distilled water

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)

6X loading dye + SDS solution (Fermentas)

Micellular casein powder from CCL stock (for positive control) Gel box and power supply from BioCurious

Simply Blue Safe Stain LC6060 Lot#1540663

Results:

   Preparation of casein control
       Take 500ul of distilled water in eppendorf tube + few grains of casein powder (use 200ul hp). Vortex vigorously.
       
   
   Preparation of samples to load on gel
       Take 40ul of 1, 8, 3B, 6B, +7F also the negative control to eppendorf tubes
       5ul of 6x loading dye to each tube (this isn't to final conc. of 1x; should be 8ul)
       Boil all the tubes for 10 mins at 95C (in order to denature the protein structure to linear) 
   Loading & Running Gel Generally, followed manufacturer's protocol for running Tris-Glycine 4%-20% mini-gels, Media:LifeTech_NuPAGE_TrisGlycine_Mini_Gels_Cat-EC6025BOX.pdf‎