Yu:CoIP: Difference between revisions

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m (Altered TEV for eppendorf tubes)
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#Incubate slowly rotating at 4°C for 2h to allow ProteinA binding.
#Incubate slowly rotating at 4°C for 2h to allow ProteinA binding.
#Spin down at 3K RPM for 30s and transfer supernatant into new tubes. Save these tubes as '''Supernatant Control'''.
#Spin down at 3K RPM for 30s and transfer supernatant into new tubes. Save these tubes as '''Supernatant Control'''.
#*For TAP mini-purification, go to [[Yu:CoIP#TEV_Cleavage|TEV Cleavage]]. Otherwise continue.
#Wash three times with 1 mL completed lysis buffer. Mix very gently by slow inversion each time, and spin down at 3k RPM for 30s.
#Wash three times with 1 mL completed lysis buffer. Mix very gently by slow inversion each time, and spin down at 3k RPM for 30s.
#*For TAP mini-purification, go to [[Yu:CoIP#TEV_Cleavage|TEV Cleavage]]. Otherwise continue.
#Wash with 300 µL 5 mM ammonium acetate, pH 5.0. Save 10 μL from each sample as '''Wash Control''', discard the remaining supernatant.
#Wash with 300 µL 5 mM ammonium acetate, pH 5.0. Save 10 μL from each sample as '''Wash Control''', discard the remaining supernatant.
#Elute with 300 µL 0.5 M acetic acid, pH 3.5. Briefly vortex after adding acetic acid, spin down, and transfer supernatant to a new tube labeled '''Eluate'''.
#Elute with 300 µL 0.5 M acetic acid, pH 3.5. Briefly vortex after adding acetic acid, spin down, and transfer supernatant to a new tube labeled '''Eluate'''.
Line 46: Line 46:
===TEV Cleavage===
===TEV Cleavage===


#Transfer beads to mobicols (small columns) in completed lysis buffer.
#Wash IgG beads twice with 1mL completed lysis buffer. Mix gently by slow inversion each time, and spin down at 2.5k RPM for 30s.
#Spin down for 2 minutes at 2k RPM.
#Wash once with with 1mL completed [[Yu:IPP|IPP150]].
#Wash with 0.5 ml [[Yu:TEV_Cleavage_Buffer|TEV Buffer]] (without DTT).   
#Wash once with 0.5 ml [[Yu:TEV_Cleavage_Buffer|TEV Buffer]] (without DTT).   
#Spin down for 2 minutes at 2.5k RPM.
#Add 100 µl TEV buffer (with DTT) + 1 µl TEV.
#Re-plug the column, and add 100 µl TEV buffer (with DTT) + 1 µl TEV.
#Incubate rotating at 16°C for 90 minutes.
#Incubate rotating at 16°C for 90 minutes.
#Spin down for 2 minutes at 2.5k RPM. Transfer the supernatant to a new tube labeled '''Eluate'''. Add 27 μL 100% TCA and freeze at -20°C overnight.
#Spin down for 30 seconds at 2.5k RPM. Transfer the supernatant to a new tube labeled '''TEV Eluate'''.
#Re-plug, and add 300 µl 0.5 M acetic acid, pH 3.5. Vortex briefly, spin down, and transfer supernatant to a new tube labeled '''TEV Control'''. Add 66μL 100% TCA and freeze at -20°C overnight.
#Add 100μL TEV buffer, gently resuspend beads, spin down at 2.5k RPM for 30s, and add the supernatant to '''TEV Eluate'''. Add 52μL 100% TCA to Eluate and freeze overnight at -20°C.
#Add 300 µl 0.5 M acetic acid, pH 3.5. Vortex briefly, spin down, and transfer supernatant to a new tube labeled '''TEV Control'''. Add 66μL 100% TCA and freeze at -20°C overnight.


==Precipitating Protein==
==Precipitating Protein==

Revision as of 09:44, 16 April 2010

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Lysis

FastPrep

  1. Prepare cell pellets.
    1. Grow cell culture to late-log overnight in 5 mL YEPD.
    2. Subclone at 1:100 into 50 mL YEPD and grow until mid-log. This takes approximately 4-6 hours.
    3. Harvest cells and transfer into a FastPrep tube.
  2. Add 100µL completed lysis buffer (PBSMT or NP-40) and approximately 100 µL glass beads. Fastprep twice for 30s at 6.5 each time. Add an additional 500 µL completed lysis buffer and spin down at max speed for 10 minutes.
  3. Transfer supernatant to a fresh eppendorf tube without disturbing pelleted beads.
  4. Perform a Bradford assay on each lysate to determine protein concentration.
  5. Transfer 5 mg protein from each lysate to a fresh eppendorf tube and bring the volume to 1 mL with completed lysis buffer.
  6. Save 10 µL of each diluted lysate as Lysate Control in new tubes.

Preparing Beads

IgG Beads

  1. Thoroughly mix IgG beads in stock bottle by swirling. Open carefully.
  2. Pipette (carefully and slowly) 35 µL of bead slurry into separate eppendorf tubes for each sample. Remix bottle after each aliquot.
  3. Add 1.5 mL TST buffer to each tube, resuspending beads by inversion.
  4. Spin down at 3K rpm for 30s. Carefully pipette off supernatant without disturbing bead pellet.
  5. Wash with 450 µL of 0.5 M acetic acid (pH 3.5). Resuspend beads by inversion and spin down again. Carefully discard supernatant.
  6. Wash with 1.5 mL TST buffer.
  7. Wash with 450 µL of 0.5 M acetic acid (pH 3.5).
  8. Wash twice, using 1.5 mL TST buffer each time.
  9. Add 1.5 ml TST, resuspend beads, and spin down. Test the pH of the supernatant with pH paper. If the pH of the supernatant is below pH 7, discard the supernatant and repeat this step to rewash the beads with TST.
  10. If the pH is 7, discard supernatant and resuspend beads in 55 µL of lysis buffer.

Isolating Protein

TAP Binding

  1. For each sample, add 1mL of 5 mg/mL protein sample to a tube containing washed IgG beads
  2. Incubate slowly rotating at 4°C for 2h to allow ProteinA binding.
  3. Spin down at 3K RPM for 30s and transfer supernatant into new tubes. Save these tubes as Supernatant Control.
    • For TAP mini-purification, go to TEV Cleavage. Otherwise continue.
  4. Wash three times with 1 mL completed lysis buffer. Mix very gently by slow inversion each time, and spin down at 3k RPM for 30s.
  5. Wash with 300 µL 5 mM ammonium acetate, pH 5.0. Save 10 μL from each sample as Wash Control, discard the remaining supernatant.
  6. Elute with 300 µL 0.5 M acetic acid, pH 3.5. Briefly vortex after adding acetic acid, spin down, and transfer supernatant to a new tube labeled Eluate.
  7. Add 20 µL 100% TCA.
  8. Freeze at -20°C overnight.

TEV Cleavage

  1. Wash IgG beads twice with 1mL completed lysis buffer. Mix gently by slow inversion each time, and spin down at 2.5k RPM for 30s.
  2. Wash once with with 1mL completed IPP150.
  3. Wash once with 0.5 ml TEV Buffer (without DTT).
  4. Add 100 µl TEV buffer (with DTT) + 1 µl TEV.
  5. Incubate rotating at 16°C for 90 minutes.
  6. Spin down for 30 seconds at 2.5k RPM. Transfer the supernatant to a new tube labeled TEV Eluate.
  7. Add 100μL TEV buffer, gently resuspend beads, spin down at 2.5k RPM for 30s, and add the supernatant to TEV Eluate. Add 52μL 100% TCA to Eluate and freeze overnight at -20°C.
  8. Add 300 µl 0.5 M acetic acid, pH 3.5. Vortex briefly, spin down, and transfer supernatant to a new tube labeled TEV Control. Add 66μL 100% TCA and freeze at -20°C overnight.

Precipitating Protein

  1. Remove eluates from -20°C and thaw on ice. Vortex thoroughly after thawed.
  2. Spin down for 15 minutes at 13k RPM at 4°C.
  3. Carefully, without disturbing the protein pellet, draw off and discard supernatant.
  4. Add 200μL ice-cold acetone (store at -20°C).
  5. Spin down for 15 minutes at 13k RPM at 4°C.
  6. Carefully draw off acetone. Leave ≈10μL in the tube so that the protein pellet is undisturbed.
  7. Evaporate remaining acetone by drying in a fume or biosafety hood.
  8. Once completely dry, add 15μL of 1x SDS-PAGE gel loading buffer and boil at 100°C for 5 minutes.
  9. Add 1μL Tris pH 8.8 if gel loading buffer dye turns yellow (indicating low pH).
  10. Run eluates on an SDS-PAGE Gel for Western blot or silver staining.
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