keronha.blogg.se - Licor western blot

Scan using licor for total protein, which will be used to normalize the blot.

Rinse twice in revert wash solution (60ml MeOH, 13.4 ml Acetic Acid, 126.6 ml Water).

Stain for total protein with Revert total protein stain on rocker for 5 minutes -when finished pour total protein stain back in bottle for later use!.

Transfer 4h at 75V (in cold room) or overnight at 35V (room temp with an ice pack).

Place in apparatus so that the black sandwich touches the black transfer piece.

Make sandwich (black side, sponge, filter paper, gel, nitrocellulose, filter paper, sponge, clear side), ensuring no bubbles between layers with black piece on bottom and layer as above.

(Tip: Gel runs more evenly if you start a lower V and increase once the samples have run down 1/3 of the gel.) Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel.Load 3 microliters of protein ladder (purple top) (in the 4 degree), and 10 microliters of each sample into separate wells.Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back Remove it from the packaging, remove the white strip of tape from the bottom back, and gently pull the comb out and rinse with water. Use a prepared 4-12% tris gel (in the 4 degree).Run SDS-PAGE gel using SDS-PAGE Running Buffer and prepare diluted transfer buffer.Transfer Apparatus, either Bio-Rad or Invitrogen.Transfer Buffer (200 mL Methanol, 100 mL 10X Transfer Buffer to final 1L volume).