⚗️PROTEIN PURIFICATION

This protocol describes the batch purification of recombinant His-tagged proteins expressed in E. coli. Proteins containing a 6xHis tag are purified using Ni-NTA (Nickel-Nitrilotriacetic Acid) beads.

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🧪 Batch Purification of His-tagged Proteins

Abstract

The His-tag enables selective binding to nickel ions, allowing efficient purification under native conditions. After cell lysis, the lysate is incubated with Ni-NTA beads, followed by washing and elution using imidazole.

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⚡ Before Starting

We recommend streaking a sample on both an autoinduction plate and a non-induced control plate to assess specificity and expression efficiency.

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🧬 Cell Lysis

1. Harvest the induced biomass using a sterile microscope slide, and resuspend it in 12 mL of lysis buffer.

Table 1. Lysis Buffer Composition

Component	Final Concentration
PBS (1X)	—
Triton X-100	1% (v/v)

1. Sonicate the suspension using a 650 W ultrasonic processor, with the following cycle:

   • 1 cycle of 5 min, 3 seconds ON / 4 seconds OFF

   • 70% amplitude, using a 6 mm probe

   • Keep on ice bath. Repeat until lysate becomes transparent.

2. Centrifuge at 15,000 g for 15 min.

3. Collect the supernatant for purification.

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🌍 Day 1 – Binding to Ni-NTA Beads

1. Transfer 50 µL of Ni-NTA beads slurry into a 1.5 mL Eppendorf tube.

2. Equilibrate beads:

   • Add 1 mL of binding buffer, rotate 5 min, centrifuge at 10,000 rpm for 5 min.

   • Discard supernatant.

   • Repeat once more with fresh binding buffer.

3. Add supernatant from sonication to the washed beads and incubate overnight at 4°C in a rotating mixer.

Table 2. Binding Buffer (pH 7.5)

Component	Final Concentration
Tris-HCl	50 mM
NaCl	500 mM
Imidazole	10 mM

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🚜 Day 2 – Washing and Elution

1. Centrifuge beads at 10,000 rpm for 5 min, collect the flowthrough and keep it on ice.

2. Wash beads twice with 250 µL (5× bead volume) of wash buffer:

   • Rotate for 5 min, centrifuge 5 min.

   • Keep Wash 1 and Wash 2 on ice.

Table 3. Low-Imidazole Wash Buffer (pH 7.5)

Component	Volume (250 µL)	Volume (1 mL)
Tris-HCl (50 mM) + NaCl (500 mM)	249.75 µL	999.5 µL
Imidazole (1 M stock)	0.25 µL	0.5 µL
Final Imidazole	6 mM	6 mM

1. Elute the protein:

   • Add 300 µL of elution buffer (6× bead volume), rotate 5 min, centrifuge 5 min.

   • Collect Elution 1.

2. Repeat elution with another 300 µL, collect as Elution 2.

Table 4. Elution Buffer (pH 7.5)

Component	Volume (300 µL)
Tris-HCl (50 mM) + NaCl	296.25 µL
Imidazole (1 M stock)	3.75 µL
Final Imidazole	300 mM

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🥣 Bead Cleaning and Storage

1. Wash beads 3 times with distilled water, rotating 1 hour each time. Centrifuge and discard supernatant.

2. Resuspend beads in equal volume of 20% ethanol.

3. Store at 4°C.

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💪 SDS-PAGE Sample Preparation

1. Prepare samples:

• Flowthrough: 5 µL sample + 5 µL H2O + 3.3 µL SB 4X

• Washes/Elutions: 10 µL sample + 3.3 µL SB 4X

• Boil at 95°C for 10 minutes.

1. Run on polyacrylamide gel:

• 10–15% resolving gel + 4% stacking gel.

• Include non-induced controls for comparison.

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🎯 Applications

This purification method has been used successfully to purify:

• BST LF mCherry protein

• Open Vent mCherry protein

• PFU DNA polymerase

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