Q1: In the E. coli protein expression system, can proteins be purified from either the soluble or insoluble fraction of the cell lysate?

A1: We can purify proteins from both fractions. The choice will depend on the customer’s experimental requirements. Our scientists have extensive experience in protein renaturation. The selection of a specific renaturation strategy is based on the protein’s sequence and structural characteristics.

Q2: If an affinity tag is used, can it be cleaved from the protein after purification?

A2: Yes, we use tags that can be cleaved off and removed from the purified protein after purification (for example, by introducing thrombin or TEV protease cleavage sequences during gene synthesis—some amino acids may remain on the protein after cleavage).

Q3: After immobilizing the antibody, the first round of affinity purification of the protein was successful, but the protein failed to bind to the antibody during the second round of purification. What is the reason for this?

A3: Low-pH elution is the most commonly used elution method in affinity purification, but it can cause denaturation of certain antibodies, which can negatively affect subsequent antigen binding. To prevent this, immediately wash the column with a neutralizing or binding buffer after protein purification, and store it in a buffer containing an antimicrobial agent such as sodium azide at 4°C. Alternatively, use a near-neutral, high-salt elution buffer, such as Pierce Gentle Ag/Ab Elution Buffer.

Q4: What method should be used to purify antibodies containing cysteine?

A4: SulfoLink Coupling Resin or UltraLink Iodoacetyl Resin are good choices, as they utilize the same chemical reaction to bind to reduced thiol groups.

Q5: How does biotin affinity purification work?

A5: Immobilized avidin, streptavidin, or neutral avidin are used to purify biotinylated molecules. The high affinity of these molecules for biotin requires irreversible denaturation to release the biotin itself (including boiling in SDS-PAGE sample buffer or 8 M guanidine, pH 1.5).

Q6: Can you provide some recommendations regarding protein refolding after purification?

A6: Please review the following recommendations:

* Maintain a low protein concentration of 10–50 μg/mL.

* Disulfide bonds help stabilize native proteins; add a redox pair, such as GSH (reduced glutathione).

* GSSG (oxidized glutathione) at a ratio of 10:1, with a GSH concentration of 2–5 mM. This redox pair helps generate the oxidative potential needed to break and form new S–S bonds during the folding process.

* Slowly remove denaturants by dilution or dialysis; glycine (50 mM, pH 9.0, 5 mM EDTA) aids in protein solubilization. If guanidine hydrochloride (GuHCl) is used, add 2 M urea, as urea also helps stabilize protein folding.

* Add detergents at very low concentrations, such as 0.1–0.5% NP-40 or 0.005% (v/v) Tween 20.

* Include cosolvent to stabilize the protein, such as glycerol (5–20%), PEG 8000, glucose, or sucrose (10%).

* Certain anionic (e.g., phosphate or sulfate) or cationic (e.g., MES or HEPES) salts also have a beneficial effect; include salts and maintain a neutral pH, such as 100 mM KCl, or 150–500 mM NaCl, and 2 mM MgCl₂.

* Protein degradation can be prevented by adding protease inhibitors such as 0.5 mM PMSF, 0.005–2 g/mL aprotinin, 2 g/mL aprotinin, or 2–5 g/mL leucinylpeptidase.

* Dialysis in phosphate buffer in the presence of calcium will result in calcium phosphate precipitation. If subsequent enterokinase digestion requires CaCl₂ (10 mM Tris, pH 8; 10 mM CaCl₂), be sure to add CaCl₂ after dialysis is complete.

Q7: How many purification runs can be performed using the same affinity column?

A7: This varies depending on the stability of the immobilized protein and the type of elution buffer used; typically, the column can be reused at least 10 times without significant loss of activity.

KMD Bioscience is renowned in the biotechnology field for its exceptional service and technical expertise, particularly in protein purification services. We offer comprehensive protein purification services designed to support the complex needs of the research community and the biopharmaceutical industry. Utilizing the latest purification technologies and methods—including affinity chromatography, ion-exchange chromatography, gel filtration chromatography, and other advanced techniques—KMD Bioscience is able to efficiently purify a wide variety of proteins, including recombinant proteins, antibodies, enzymes, and other bioactive molecules. Our services begin with the selection and optimization of expression systems, proceed through protein expression, lysis, and preliminary purification, and culminate in the fine purification and quality assessment of the final product, with every step adhering to strict quality control standards. In addition, the KMD Bioscience team provides personalized consulting to help clients address specific challenges that may arise during the purification process, ensuring that the final product meets the expected purity, activity, and stability. Whether for basic research, drug development, or other industrial applications, we are committed to providing efficient and reliable protein purification solutions to accelerate the translation and application of scientific research findings.