Difference between revisions of "Immunoprecipitation"
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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) | ||
Latest revision as of 10:24, 2 April 2014
Immunoprecipitation Protocol by Adam Kimbrough from Laboratory of Dr. Debi Fadool
- Prepare lysis buffer on ice
- 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
- Harvest fresh tissue
- Homogenize 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
- use a siliconized pipette to transfer homogenized tissue to tubes
- 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
- Remove supernatant to fresh tube and save pellet in case it contains protein
- Fraction supernatant
- 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 remainder for IP pre-clear split into tubes for equal amounts per IP antibody
- Pre-clear with protein A sephirose beads
- Reconstitute sephirose into a homogenious mixture by raking against ependorf tube rack and shaking
- Cut pipette tip for sucking up of sephirose
- 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
- 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
- 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)
- IP overnight
- Add appropriate amount of pre-clear soluation to tubes for equal amounts of protein as calculated by Bradford assay (see step 9)
- 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
- finish IP the following day
- Add protein A sephirose beads for 3 hours
- Reconstitute as above
- 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)
- 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)
- 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
- Samples are now ready for running on Western Blots
- use 15 ul of each sample per run (2 runs/sample per IP)