- Brand: ELEM粒曼
- Commodity name: Strep-tag purification resin
- Commodity ID: LMMS04004
- 名称: Strep-tag purification resin
- 数量: 5ml
- 保存条件: 4°C, 20% ethanol, or PBS containing 0.5% Na₂N₂ at 4°C
Strep-tag purification resin is a bioaffinity chromatography medium prepared by covalently immobilizing Strep‑tag protein onto agarose gel beads, primarily used for the purification of Strep‑tagged proteins. After binding to the resin, these proteins can be eluted using a competitive desulfation–biotin approach, which is mild and typically does not compromise the protein’s integrity. If the protein is tolerant to moderate alkalinity, an alkaline elution buffer—such as 10 mM NaOH—may also be employed. For quality control of the purification resin and optimization of experimental conditions, we recommend using GenScript’s EGFP‑StrepII‑tagged protein (Catalog No. LMMS04005).
1. Sample Preparation:

01. Protein induction expression: Express strep-tagged protein in cells.

02. Resuspend the cells in binding buffer; alternatively, use sonication for cell lysis. Ensure that the sample’s binding buffer is identical to the equilibration buffer. (If necessary, add an appropriate amount of SPMF protease inhibitor.)

2. Chromatography (using 1 mL of packing material as an example)

01. Transfer 1 mL of Strep-tag purification resin into a 50-mL centrifuge tube, wash with five times the volume of binding buffer, centrifuge at 3,000 × g for 5 minutes, discard the supernatant, and repeat three times. Mix the prepared sample with the equilibrated resin and incubate at room temperature for 1 hour.

02. Load the packing into the column, collect the effluent, and record it as “eluate.”

03. Wash the packing material with 6 mL of binding buffer.

04. Wash with 2.5 mL of elution buffer (binding buffer + 2.5 mM desthiobiotin), designated as the first fraction; wash a second time with another 2.5 mL of elution buffer, designated as the second fraction. The elution volume may be increased until all the protein has been eluted.

05. Perform SDS-PAGE analysis on the sample stock solution, flow-through fraction, and eluate.

06. NaOH column regeneration: After eluting the target protein, flush the column with distilled water for 3–5 column volumes.

07. Regenerate the column with 0.5 M NaOH, using 3–5 column volumes; then wash with distilled water until neutral, and store the column in 20% ethanol.

08. HABA Regeneration: After eluting the target protein with desulfurized biotin, the column can also be regenerated using HABA buffer. Typically, wash with 15 column volumes of HABA binding buffer, followed by 30 column volumes of binding buffer. The column can then be stored in 20% ethanol or proceed directly to the next purification step. After loading onto the HABA column, the gel turns a reddish‑orange color; upon equilibration with binding buffer, it returns to its normal white appearance. This regeneration method generally requires a larger buffer volume.

3. Buffer Preparation:
- Purification of Strep II‑tagged protein:
Binding buffer: 100 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0; or 20 mM NaH₂PO₄, 280 mM NaCl, 6 mM KCl, pH 7.4

Elution buffer: Binding buffer + 2.5 mM desthiobiotin; or 10 mM NaOH

Regeneration buffer: 0.5 M NaOH; or binding buffer + 1 mM HABA
- Purification of Strep-tag–tagged proteins:
Binding buffer: 100 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0; or 20 mM NaH2PO4, 280 mM NaCl, 6 mM KCl, pH 7.4. Elution buffer: binding buffer supplemented with 5 mM desthiobiotin.

Regeneration buffer: 0.5 M NaOH; or binding buffer + 1 mM HABA

4. Detection and Storage of the Target Protein

01. Determine the distribution of the target protein in the eluate. The most effective approach is SDS/PAGE analysis. Alternatively, you can use a Bradford protein assay kit to rapidly quantify the protein concentration, followed by SDS/PAGE analysis to assess its distribution.

02. The need for further purification of the target protein should be determined based on its intended application. The storage conditions for the purified target protein should be established according to its physicochemical properties and intended use.
Precautions:
- The sample must be clarified and free of particulates; otherwise, it may clog the column and shorten its service life. Prior to loading protein samples, it is recommended to filter them through a 0.45 μm filter.
- If high protein purity is required, do not reuse the packing material. Each 1 mL of Strep‑tag purification resin can bind more than 3.5 mg of fusion protein.
- If the fusion protein is expressed as an inclusion body, it can only be purified using Strep‑tag affinity resin after renaturation. However, the typical renaturation process is time‑consuming, labor‑intensive, and often difficult to achieve success.
- The buffer solution can be prepared according to your experimental requirements; however, please perform a small-scale purification test before use.
Frequently Asked Questions:

Issue 1: The target protein does not bind to the resin, or the binding efficiency is low.

Solutions and Recommendations:

01. The target protein lacks a strep‑Tag; sequencing confirms this, the reading frame is adjusted, a protease‑deficient E. coli strain is used as the expression host, and a protease inhibitor is employed.

02. The strep-tag fusion protein is degraded upon sonication; it is recommended to use bacterial lysis buffer to gently lyse E. coli at room temperature, which ensures complete cell lysis. The supernatant can then be mixed with two volumes of equilibration buffer and directly loaded onto the column for purification.

03. The binding conditions are incorrect; please check the pH of the binding buffer and use an appropriate binding buffer.

04. The packing material has been used too many times; if it has already undergone multiple cycles, consider regeneration or replacement with new resin.

Issue 2: The protein cannot be eluted, or the elution efficiency is low.

Solutions and Recommendations:

01. If the flow rate during elution is too fast, it is recommended to block the outlet after adding the elution buffer and gently shake the column to ensure thorough contact between the buffer and the packing material, or to perform gentle shaking.

02. The concentration of the strep-tagged protein in the lysate supernatant is too low: concentrate the target supernatant to increase the target protein concentration, and optimize the expression conditions; if a strep tag is used, consider switching to a twin-strep tag to enhance binding affinity.

03. The pH of the elution buffer is incorrect.

04. If the strep-tag fusion protein has already undergone degradation and multiple bands appear upon addition of a protease inhibitor to the lysis buffer, this is likely due to partial proteolytic cleavage of the target protein. In such cases, a protease-deficient host strain (e.g., lon or ompT) should be used.

Question 3: Impurities are present in the eluted protein.

Solutions and Recommendations:

01. The sample load is too high; reduce the loading amount or increase the wash volume. Not all target proteins are full-length; if the cDNA contains rare codons, use a Rosetta‑series strain as the host.

02. Over‑sonication should be avoided; reduce sonication time to prevent foaming and protein denaturation. It is recommended to lyse E. coli gently at room temperature using a bacterial lysis buffer, which can achieve complete cell lysis. The supernatant can then be directly loaded onto the column for purification after adding two volumes of equilibration buffer.

03. Some additional bands observed on the covalent purification gel correspond to molecular chaperones that facilitate the proper folding of the target protein, such as DnaK (70 kDa), DnaJ (37 kDa), GrpE (40 kDa), GroEL (57 kDa), and GroES (10 kDa); further purification steps can help resolve these bands.

Question 4: The target protein appears in the Strep-tag protein elution buffer.

Solutions and Recommendations:

If the protein concentration in the supernatant is too high, reduce the sample load or increase the wash volume. When the wash volume is excessively large, it is normal for Strep‑tag–labeled proteins to be eluted.

None

Product Reagent List

Product Introduction

Product instructions

Other Notes

Classification： strepQ Filler

Product Reagent List

Name

Strep-tag purification resin

Quantity

5ml

Save condition

4°C, 20% ethanol, or PBS containing 0.5% Na₂N₂ at 4°C

Product Introduction

Strep-tag purification resin is a bioaffinity chromatography medium prepared by covalently immobilizing Strep‑tag protein onto agarose gel beads, primarily used for the purification of Strep‑tagged proteins. After binding to the resin, these proteins can be eluted using a competitive desulfation–biotin approach, which is mild and typically does not compromise the protein’s integrity. If the protein is tolerant to moderate alkalinity, an alkaline elution buffer—such as 10 mM NaOH—may also be employed. For quality control of the purification resin and optimization of experimental conditions, we recommend using GenScript’s EGFP‑StrepII‑tagged protein (Catalog No. LMMS04005).

Product instructions

1. Sample Preparation:

01. Protein induction expression: Express strep-tagged protein in cells.

02. Resuspend the cells in binding buffer; alternatively, use sonication for cell lysis. Ensure that the sample’s binding buffer is identical to the equilibration buffer. (If necessary, add an appropriate amount of SPMF protease inhibitor.)

2. Chromatography (using 1 mL of packing material as an example)

01. Transfer 1 mL of Strep-tag purification resin into a 50-mL centrifuge tube, wash with five times the volume of binding buffer, centrifuge at 3,000 × g for 5 minutes, discard the supernatant, and repeat three times. Mix the prepared sample with the equilibrated resin and incubate at room temperature for 1 hour.

02. Load the packing into the column, collect the effluent, and record it as “eluate.”

03. Wash the packing material with 6 mL of binding buffer.

04. Wash with 2.5 mL of elution buffer (binding buffer + 2.5 mM desthiobiotin), designated as the first fraction; wash a second time with another 2.5 mL of elution buffer, designated as the second fraction. The elution volume may be increased until all the protein has been eluted.

05. Perform SDS-PAGE analysis on the sample stock solution, flow-through fraction, and eluate.

06. NaOH column regeneration: After eluting the target protein, flush the column with distilled water for 3–5 column volumes.

07. Regenerate the column with 0.5 M NaOH, using 3–5 column volumes; then wash with distilled water until neutral, and store the column in 20% ethanol.

08. HABA Regeneration: After eluting the target protein with desulfurized biotin, the column can also be regenerated using HABA buffer. Typically, wash with 15 column volumes of HABA binding buffer, followed by 30 column volumes of binding buffer. The column can then be stored in 20% ethanol or proceed directly to the next purification step. After loading onto the HABA column, the gel turns a reddish‑orange color; upon equilibration with binding buffer, it returns to its normal white appearance. This regeneration method generally requires a larger buffer volume.

3. Buffer Preparation:

Purification of Strep II‑tagged protein:

Binding buffer: 100 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0; or 20 mM NaH₂PO₄, 280 mM NaCl, 6 mM KCl, pH 7.4

Elution buffer: Binding buffer + 2.5 mM desthiobiotin; or 10 mM NaOH

Regeneration buffer: 0.5 M NaOH; or binding buffer + 1 mM HABA

Purification of Strep-tag–tagged proteins:

Binding buffer: 100 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0; or 20 mM NaH2PO4, 280 mM NaCl, 6 mM KCl, pH 7.4. Elution buffer: binding buffer supplemented with 5 mM desthiobiotin.

Regeneration buffer: 0.5 M NaOH; or binding buffer + 1 mM HABA

4. Detection and Storage of the Target Protein

01. Determine the distribution of the target protein in the eluate. The most effective approach is SDS/PAGE analysis. Alternatively, you can use a Bradford protein assay kit to rapidly quantify the protein concentration, followed by SDS/PAGE analysis to assess its distribution.

02. The need for further purification of the target protein should be determined based on its intended application. The storage conditions for the purified target protein should be established according to its physicochemical properties and intended use.

Precautions:

The sample must be clarified and free of particulates; otherwise, it may clog the column and shorten its service life. Prior to loading protein samples, it is recommended to filter them through a 0.45 μm filter.
If high protein purity is required, do not reuse the packing material. Each 1 mL of Strep‑tag purification resin can bind more than 3.5 mg of fusion protein.
If the fusion protein is expressed as an inclusion body, it can only be purified using Strep‑tag affinity resin after renaturation. However, the typical renaturation process is time‑consuming, labor‑intensive, and often difficult to achieve success.
The buffer solution can be prepared according to your experimental requirements; however, please perform a small-scale purification test before use.

Other Notes

Frequently Asked Questions:

Issue 1: The target protein does not bind to the resin, or the binding efficiency is low.

Solutions and Recommendations:

01. The target protein lacks a strep‑Tag; sequencing confirms this, the reading frame is adjusted, a protease‑deficient E. coli strain is used as the expression host, and a protease inhibitor is employed.

02. The strep-tag fusion protein is degraded upon sonication; it is recommended to use bacterial lysis buffer to gently lyse E. coli at room temperature, which ensures complete cell lysis. The supernatant can then be mixed with two volumes of equilibration buffer and directly loaded onto the column for purification.

03. The binding conditions are incorrect; please check the pH of the binding buffer and use an appropriate binding buffer.

04. The packing material has been used too many times; if it has already undergone multiple cycles, consider regeneration or replacement with new resin.

Issue 2: The protein cannot be eluted, or the elution efficiency is low.

Solutions and Recommendations:

01. If the flow rate during elution is too fast, it is recommended to block the outlet after adding the elution buffer and gently shake the column to ensure thorough contact between the buffer and the packing material, or to perform gentle shaking.

02. The concentration of the strep-tagged protein in the lysate supernatant is too low: concentrate the target supernatant to increase the target protein concentration, and optimize the expression conditions; if a strep tag is used, consider switching to a twin-strep tag to enhance binding affinity.

03. The pH of the elution buffer is incorrect.

04. If the strep-tag fusion protein has already undergone degradation and multiple bands appear upon addition of a protease inhibitor to the lysis buffer, this is likely due to partial proteolytic cleavage of the target protein. In such cases, a protease-deficient host strain (e.g., lon or ompT) should be used.

Question 3: Impurities are present in the eluted protein.

Solutions and Recommendations:

01. The sample load is too high; reduce the loading amount or increase the wash volume. Not all target proteins are full-length; if the cDNA contains rare codons, use a Rosetta‑series strain as the host.

02. Over‑sonication should be avoided; reduce sonication time to prevent foaming and protein denaturation. It is recommended to lyse E. coli gently at room temperature using a bacterial lysis buffer, which can achieve complete cell lysis. The supernatant can then be directly loaded onto the column for purification after adding two volumes of equilibration buffer.

03. Some additional bands observed on the covalent purification gel correspond to molecular chaperones that facilitate the proper folding of the target protein, such as DnaK (70 kDa), DnaJ (37 kDa), GrpE (40 kDa), GroEL (57 kDa), and GroES (10 kDa); further purification steps can help resolve these bands.

Question 4: The target protein appears in the Strep-tag protein elution buffer.

Solutions and Recommendations:

If the protein concentration in the supernatant is too high, reduce the sample load or increase the wash volume. When the wash volume is excessively large, it is normal for Strep‑tag–labeled proteins to be eluted.

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