Splitting Cells

2018-08-30T14:11:22Z

Ayata: included instructions to make more DMEM

==Materials==
*Media (FBS or NCS as required): Prepare by filtering together 5 mL PSG, 50 mL COS-FBS and one bottle of DMEM
(To prepare DMEM:
1 bottle of DMEM found in 4c in cell culture room
1 tube FBS found in -80 in cell culture room
5 mLs PSG found in door of 4c in cell culture room
500 mL bottle taken from lab
filter found on metal rack behind fume hood
•pour all ingredients into DMEM bottle
•screw filter onto 500 mL bottle
•attach hose onto arm of filter
•pour mixture into filter)
*PBS -/-
*0.05% Trypsin

==Protocol==
#Warm PBS and Media in water bath
# Aspirate the plate media
#Wash cells once with 10 mL (per 10 cm dish) PBS -/- then aspirate the PBS
#Add 1 mL trypsin and allow to sit in the hood for 2-5 min
#Add 10 mL media to each new dish
#Check cells for trypsinization, and if necessary tap the cells
#Add 9 mL media to trypsinized cells
#Add 1 mL cells to each dish (for 10X dilution; 0.5 mL for 20X)
#Replace plates in the 37C incubator

==Cell Specific Notes==
*3T3-L1 fibroblasts have special considerations regarding confluence. See [[Differentiation of 3T3-L1 Cells]]
*RAW 264.7 cells are scraped, not trypsinized. See [[Culturing RAW 264.7 Cells]]
*S2 cells are grown at 28C without extra CO2. See [[Culturing S2 Cells]]
[[ Category:Cell Culture ]]

Western Blotting

2018-07-12T15:34:18Z

Ayata:

==Materials==
*Transfer Buffer (200 mL Methanol, 100 mL 10X [[ Transfer Buffer ]] to final 1L volume)
*Transfer Apparatus, either Bio-Rad or Invitrogen

==Protocol==
#Run SDS-PAGE gel using [[ SDS-PAGE Running Buffer ]] and prepare diluted transfer buffer
## Use a prepared 5-12% tris gel (in the 4 degree). 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.
##Load into gel tank. Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back
##Load 3 microliters of protein ladder (purple), and 10 microliters of each sample into separate wells.
## Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel
#Make sandwich, ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.
#Transfer 4h at 75V (in cold room) or overnight at 35V (room temp).
#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!
#Rinse twice in revert wash solution (60ml MeOH, 13.4 ml Aceditc Acid, 126.6 ml Water)
#Scan using licor for total protein, which will be used to normalize the blot
#Rinse nitrocellulose in revert reversal solution for at least 5 and no more than 10 minutes until nitrocellulose appears clear again (.2g NaOH, 60ml MeOH, 140ml Water)
#Rinse nitrocellulose in 2% BSA (2g BSA in 100ml TBST, stored in fridge) for 1 hour
#Incubate with primary antibody (check for dilution) in 2% BSA for >1h
#Wash blot every 5 minutes for 15 min with TBST.
#Incubate with appropriate secondary antibody (10 000X) for 45min-1h (20 ml 2% BSA 1ul of both secondary antibodies, all found in fridge Ab stocks with blue dots on top)
#Wash blot every 5 minutes for 15 min with TBST.
#Rinse once or twice with double distilled water
#Prepare ECL by mixing one volume of the black bottle solution with one volume of the white bottle solution (be careful not to cross-contaminate ECL bottles with wrong solution).
#Drain excess buffer from blot and cover with ECL for about a minute
#Drain excess ECL from blot, cover with saran wrap and expose film

==If Using LiCor==
#Start -> New -> Scan Image -> Login -> Peloquin -> Password Located in Desk -> Select Dimensions -> Start Scan

[[ Category: Western Blotting ]]

Bridges Lab Protocols - User contributions [en]

Splitting Cells

Western Blotting