1,145
edits
Changes
fixed formating
by Adam Kimbrough from Laboratory of Dr. Debi Fadool
by Adam Kimbrough from Laboratory of Dr. Debi Fadool
#Prepare lysis buffer on ice
# Prepare lysis buffer on ice
*750ul ilysis buffer into homogenizer and 750ul added to wash out after homogenization
#* 750ul ilysis buffer into homogenizer and 750ul added to wash out after homogenization
#* add 100 ul PMSF, 10 ul P, L, A protein phosphatase inhibitors per 10ml of lysis buffer
*add 100 ul PMSF, 10 ul P, L, A protein phosphatase inhibitors per 10ml of lysis buffer
# Harvest fresh tissue
# Homogenize tissue
#Harvest fresh tissue
#* 50 strokes on ice in 750 ul lysis buffer with PPIs until completely homogenized
#* add 750 ul lysis buffer to wash the homogenized tissue
#Homogenize tissue
#* use a siliconized pipette to transfer homogenized tissue to tubes
*50 strokes on ice in 750 ul lysis buffer with PPIs until completely homogenized
# Rotate in rotary for 30 min at 4 degrees Celsius to allow to lyse
# Centrifuge for 10 minutes at 14000 rpm (setting #14 on fadool centrifuge) at 4 degrees Celsius
*add 750 ul lysis buffer to wash the homogenized tissue
# Remove supernatant to fresh tube and save pellet in case it contains protein
# Fraction supernatant
*use a siliconized pipette to transfer homogenized tissue to tubes
#* Take 50 ul of supernatant per IP antibody for a lysate fraction from each sample
#* Take 20 ul per antibody for Bradford assay from each sample
#Rotate in rotary for 30 min at 4 degrees Celsius to allow to lyse
#* Take remainder for IP pre-clear split into tubes for equal amounts per IP antibody
# Pre-clear with protein A sephirose beads
#Centrifuge for 10 minutes at 14000 rpm (setting #14 on fadool centrifuge) at 4 degrees Celsius
#* Reconstitute sephirose into a homogenious mixture by raking against ependorf tube rack and shaking
#* Cut pipette tip for sucking up of sephirose
#Remove supernatant to fresh tube and save pellet in case it contains protein
#* Add 30 ul sephirose A protein beads to each sample (i.e. 30 ul in HDAC and 30 ul in 14-3-3 tube)
#* Rotary for 1 hour minimum (can be longer) at 4 degrees Celsius
#Fraction supernatant
# Run Bradford assay and calculate amount for equal protein per sample into the IP (fractions should have same levels of protein but samples from different animals shouldn’t)
#* Calculate sample protein levels for volume to add for IP
*Take 50 ul of supernatant per IP antibody for a lysate fraction from each sample
# Centrifuge for 10 minutes at 14000 rpm at 4 degrees Celsius
# Save supernatant from pre-clear into new tube (beads[pellet at bottom] collected the antigens present to discard)
*Take 20 ul per antibody for Bradford assay from each sample
# IP overnight
#* Add appropriate amount of pre-clear soluation to tubes for equal amounts of protein as calculated by Bradford assay (see step 9)
*Take remainder for IP pre-clear split into tubes for equal amounts per IP antibody
#* 7.5 ul neat antibody (for HDAC and 14-3-3, calculate for others) for ~750 ul of IP fraction per sample
#* rotary at 4 degrees Celsius overnight with antibody in each tube
#Pre-clear with protein A sephirose beads
#* finish IP the following day
*Reconstitute sephirose into a homogenious mixture by raking against ependorf tube rack and shaking
# Add protein A sephirose beads for 3 hours
#* Reconstitute as above
*Cut pipette tip for sucking up of sephirose
#* Rotary in 4 degrees Celsius for minimum 3 hours to collect IP product to beads
# Centrifuge for 10 minutes at 14000 rpm (NOW SAVING PELLET-has IP product)
*Add 30 ul sephirose A protein beads to each sample (i.e. 30 ul in HDAC and 30 ul in 14-3-3 tube)
#* Siphon off supernatant and rinse with Wash buffer
# Repeat centrifugation for 10 minutes and wash with wash buffer 3 times (can leave small amount of liquid)
*Rotary for 1 hour minimum (can be longer) at 4 degrees Celsius
# Final rinse remove all liquid and leave only pellet
#* Add 30 ul of KFL to each sample and store at -20 degrees Celsius as long as needed
#Run Bradford assay and calculate amount for equal protein per sample into the IP (fractions should have same levels of protein but samples from different animals shouldn’t)
#Samples are now ready for running on Western Blots
Calculate sample protein levels for volume to add for IP
#* use 15 ul of each sample per run (2 runs/sample per IP)
Add appropriate amount of pre-clear soluation to tubes for equal amounts of protein as calculated by Bradford assay (see step 9)
rotary at 4 degrees Celsius overnight with antibody in each tube
finish IP the following day
Reconstitute as above
Rotary in 4 degrees Celsius for minimum 3 hours to collect IP product to beads
Siphon off supernatant and rinse with Wash buffer
Add 30 ul of KFL to each sample and store at -20 degrees Celsius as long as needed
use 15 ul of each sample per run (2 runs/sample per IP)