Changes

by  Adam Kimbrough from Laboratory of Dr. Debi Fadool

by  Adam Kimbrough from Laboratory of Dr. Debi Fadool

1. prepare lysis buffer on ice

#Prepare lysis buffer on ice

a. 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

b. 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

2. harvest fresh tissue

#Harvest fresh tissue

3. homogenize tissue

#Homogenize tissue

50 strokes on ice in 750 ul lysis buffer with PPIs until completely homogenized

*50 strokes on ice in 750 ul lysis buffer with PPIs until completely homogenized

add 750 ul lysis buffer to wash the homogenized tissue

*add 750 ul lysis buffer to wash the homogenized tissue

use a siliconized pipette to transfer homogenized tissue to tubes

#Rotate in rotary for 30 min at 4 degrees Celsius to allow to lyse

4. 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

5. Centrifuge for 10 minutes at 14000 rpm (setting #14 on fadool centrifuge) at 4 degrees Celsius

#Remove supernatant to fresh tube and save pellet in case it contains protein

6. Remove supernatant to fresh tube and save pellet in case it contains protein

#Fraction supernatant

7. Fraction supernatant

*Take 50 ul of supernatant per IP antibody for a lysate fraction from each sample

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

Take 20 ul per antibody for Bradford assay from each sample

*Take remainder for IP pre-clear split into tubes for equal amounts per IP antibody

Take remainder for IP pre-clear split into tubes for equal amounts per IP antibody

*Cut pipette tip for sucking up of sephirose

8. Pre-clear with protein A sephirose beads

*Add 30 ul sephirose A protein beads to each sample (i.e. 30 ul in HDAC and 30 ul in 14-3-3 tube)

Reconstitute sephirose into a homogenious mixture by raking against ependorf tube rack and shaking

Cut pipette tip for sucking up of sephirose

*Rotary for 1 hour minimum (can be longer) at 4 degrees Celsius

#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)

 

 

9. 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

Calculate sample protein levels for volume to add for IP