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Finkelstein Lab Wiki

Cas13d (SNAP-EsCas13d) Purification

Used in our papers:12

Add DTT before use.

Contents

Section titled “Contents”
  1. Buffers
    1. Lysis Buffer
    2. Wash Buffer
    3. Elution Buffer
    4. SUMO Proteolysis Buffer
    5. SP buffer A
    6. SP buffer B
    7. S200 Buffer
    8. Filtered ddH2O
  2. Purification
    1. Day0
    2. Day1
    3. Day2

1. Buffers

Section titled “1. Buffers”
  • Filter all buffer with 0.22uM filter and store at 4C.

1.1 Lysis Buffer (500 mL, pH 7.4)

Section titled “1.1 Lysis Buffer (500 mL, pH 7.4)”
  • 50 mM HEPES - 25 mL (1M HEPES pH 7.4)
  • 500 mM NaCl - 50 mL (5M NaCl)
  • 1 mM EDTA - 1 mL (0.5M EDTA pH 8.0)
  • 5% glycerol - 25 mL (100% glycerol)
  • 0.1% Tween-20 - 5 mL (10% Tween-20)
  • Add to 50mL lysis buffer before use:
    • 1 mM DTT - 0.5 mL (1M DTT)
    • 1 tablet of complete™ edta-free protease inhibitor cocktail.
    • 1 mg/mL lysozyme
    • 2.5 U/mL DNaseI
    • 2.5 U/mL salt active nuclease

1.2 Wash Buffer (500 mL, pH 7.4)

Section titled “1.2 Wash Buffer (500 mL, pH 7.4)”
  • 50 mM HEPES - 25 mL (1M HEPES pH 7.4)
  • 500 mM NaCl - 50 mL (5M NaCl)
  • 5% glycerol - 25 mL (100% glycerol)
  • 1mM DTT (Add DTT before use.)

1.3 Elution Buffer (30 mL, pH 7.4)

Section titled “1.3 Elution Buffer (30 mL, pH 7.4)”
  • 50 mM HEPES - mL (1M HEPES pH 7.4)
  • 500 mM NaCl - mL (5M NaCl)
  • 10% glycerol - 25 mL (100% glycerol)
  • 10 mM D-desthiobiotin
  • 1mM DTT (Add DTT before use.)

1.4 SUMO Proteolysis Buffer (1 L, pH 7.4)

Section titled “1.4 SUMO Proteolysis Buffer (1 L, pH 7.4)”
  • 50 mM HEPES - 25 mL (1M HEPES pH 7.4)
  • 100 mM NaCl - 50 mL (5M NaCl)
  • 10% glycerol - 25 mL (100% glycerol)
  • 1mM DTT (Add DTT before use.)

1.5 SP buffer A (500 mL, pH 7.4)

Section titled “1.5 SP buffer A (500 mL, pH 7.4)”
  • 50 mM HEPES - 25 mL (1M HEPES pH 7.4)
  • 100 mM NaCl - 50 mL (5M NaCl)
  • 10% glycerol - 25 mL (100% glycerol)
  • 2mM DTT (Add DTT before use.)

1.6 SP buffer B (500 mL, pH 7.4)

Section titled “1.6 SP buffer B (500 mL, pH 7.4)”
  • 50 mM HEPES - 25 mL (1M HEPES pH 7.4)
  • 1 M NaCl - 50 mL (5M NaCl)
  • 10% glycerol - 25 mL (100% glycerol)
  • 2mM DTT (Add DTT before use.)

1.7 S200 Buffer (1L, pH 8.0)

Section titled “1.7 S200 Buffer (1L, pH 8.0)”
  • 50 mM Tris - 25 mL (1M Tris pH 7.5)
  • 500 mM NaCl - 50 mL (5M NaCl)
  • 10% glycerol - 25 mL (100% glycerol)
  • 2mM DTT (Add DTT before use.)

1.8 Filtered ddH2O (500 mL)

Section titled “1.8 Filtered ddH2O (500 mL)”
  • Keep cold in 4°C
  • Mainly used to rinse off FPLC tubes before transferring from high salt buffer to water. This is important since the salt is so concentrated.

2. Purification

Section titled “2. Purification”

under construction

2.1 Day0

Section titled “2.1 Day0”

2.2 Day1

Section titled “2.2 Day1”

2.3 Day2

Section titled “2.3 Day2”

Footnotes

Section titled “Footnotes”

  1. Kuo et al. (2024). Massively Parallel Profiling of RNA-targeting CRISPR-Cas13d. Nature Communications. ↩

  2. Chou et al. (2026). Structural basis for target discrimination and activation by Cas13d. Science Advances. ↩