> For the complete documentation index, see [llms.txt](https://reclone-latam-uncuyo.gitbook.io/building-a-reagent-collaborative-network-in-latam/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://reclone-latam-uncuyo.gitbook.io/building-a-reagent-collaborative-network-in-latam/protein-purification.md).

# 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](https://dx.doi.org/10.17504/protocols.io.kqdg3wz8ev25/v2)

### 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)            |

2. 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.
3. Centrifuge at **15,000 g for 15 min**.
4. Collect the **supernatant** for purification.

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### 🌍 Day 1 – Binding to Ni-NTA Beads

5. Transfer **50 µL** of **Ni-NTA beads slurry** into a **1.5 mL Eppendorf tube**.
6. **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.
7. 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

8. Centrifuge beads at **10,000 rpm for 5 min**, collect the **flowthrough** and keep it on ice.
9. 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**      |

10. Elute the protein:
    * Add **300 µL of elution buffer** (6× bead volume), rotate 5 min, centrifuge 5 min.
    * Collect **Elution 1**.
11. 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

12. Wash beads **3 times** with **distilled water**, rotating 1 hour each time. Centrifuge and discard supernatant.
13. Resuspend beads in equal volume of **20% ethanol**.
14. Store at **4°C**.

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### 💪 SDS-PAGE Sample Preparation

15. 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**.

16. 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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