Difference between revisions of "Western Blot of bovine casein"

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'''Material:''' for SDS page:
'''Material:''' for SDS page:
SDS (10%) catalog number: SDS10-100 ,
 
SDS (10%) catalog number: SDS10-100
 
distilled water
distilled water
For western blot:
 
Gel loading tips (BioRad)
Tris-Glycine SDS Running Buffer 10X stock (Novex Life Tech, Catalog number: LC2675)
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)
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)
Protein ladder (10-250 kDa) (NEB, Catalog number: p7703S)
6X loading dye + SDS solution (Fermentas)
6X loading dye + SDS solution (Fermentas)
Micellular casein powder from CCL stock (for positive control)
Micellular casein powder from CCL stock (for positive control)
Gel box and power supply from BioCurious
Simply Blue Safe Stain LC6060 Lot#1540663






'''Results:'''
'''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

Latest revision as of 01:06, 3 May 2015

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