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fixed formating
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− 10. Centrifuge for 10 minutes at 14000 rpm at 4 degrees Celsius
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− 11. Save supernatant from pre-clear into new tube (beads[pellet at bottom] collected the antigens present to discard)
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−12. IP overnight
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− 7.5 ul neat antibody (for HDAC and 14-3-3, calculate for others) for ~750 ul of IP fraction per sample
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−13. Add protein A sephirose beads for 3 hours
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−14. Centrifuge for 10 minutes at 14000 rpm (NOW SAVING PELLET-has IP product)
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− 15. Repeat centrifugation for 10 minutes and wash with wash buffer 3 times (can leave small amount of liquid)
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−16. Final rinse remove all liquid and leave only pellet
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− 17. Samples are now ready for running on Western Blots
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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)