KMD Bioscience Co., Ltd.
400-621-6806
2018-12-13 Hits(841)
Q1: In E. coli protein expression system, will you purify the protein from the soluble fraction or insoluble fraction of the cell lysate? Can you refold insoluble protein?
A1: We can purify the protein from both fractions. It will depend on client's requirement or available protocol. Our scientists have very good experience in protein refolding. Selection of particular refolding strategy is based on proteins' sequences and their structural properties.
Q2: If you use an affinity tag, can you cleave it off the protein after purification?
A2: Yes, we use tags that can be cleaved off after purification and removed from the purified protein (for example, by introducing a thrombin or TEV protease cleavage sequence during gene synthesis-some amino acids may be left on the protein after cleavage).
Q3: After immobilizing my antibody, the first affinity purification of my protein worked well, but the protein failed to bind to the antibody during the second round of purification. What did I do wrong?
A3: Low pH elution is the most commonly used method of elution for affinity purification, however, it could lead to denaturation of some antibodies which will negatively affect subsequent antigen binding. To prevent this from happening, after protein purification and column regeneration, immediately wash the column with neutralization or binding buffer and store at 4 degrees C in buffer containing an antimicrobial agent such as sodium azide. Alternatively, use a near-neutral, high salt elution buffer such as Pierce Gentle Ag/Ab Elution Buffer.
Q4: I want to purify an antibody against a peptide that has a cysteine moiety. What support should I use?
A4: SulfoLink Coupling Resin or UltraLink Iodoacetyl Resin would be good choices. They utilize the same chemistry to react with the reduced thiol group.
Q5: How does biotin affinity purification work?
A5: Immobilized avidin, streptavidin or Neutravidin is used to purify biotinlyated molecules with high stringency. The high affinity of these molecules for biotin requires non-reversible denaturation to release biotin itself (including boiling in SDS-PAGE sample buffer or 8M guanidine, pH 1.5).
Q6: Can you provide suggestions on how to refold protein following purification?
A6: Please read our suggestions below:
-Maintain low protein concentrations 10-50µg/mL.
-Disulfide bonds help to stabilize native proteins; add a redox pair such as GSH (reduced glutathione)
-GSSG (oxidized glutathione) at a ratio of 10:1 with GSH concentration of 2-5mM. Such a redox pair is helpful to create an oxidizing potential to break and build new S-S bonds during the folding process.
-Remove denaturants slowly by dilution or dialysis; glycine (50mM, pH 9.0, 5mM EDTA) helps to solubilize the proteins. If GuHCl (guanidinium HCl) is used, add 2 M urea because urea helps to stabilize the protein folding, too.
-Add detergents at a very low concentration such as 0.1-0.5% NP-40 or 0.005% (v/v) Tween 20.
-Include co-solvents to stabilize the proteins such as glycerol (5-20%) or PEG 8000 or glucose or sucrose(10%).
-Certain anions (e.g., phosphate or sulfate) or cations (e.g., MES or HEPES) have positive effects, too; include salt and maintain a neutral pH such as 100mM KCl, or 150-500mM NaCl, 2mM MgCl2.
-Avoid protein degradation by adding protease inhibitors such as 0.5mM PMSF, 0.005-2µg/mL aprotinin, 2µg/mL Pepstatin, or 2-5µg/mL leupeptin.
-Dialysis of a phosphate buffer when against calcium will result in a calcium phosphate precipitate. If CaCl2 is needed for subsequent enterokinase digestion (10mM Tris pH 8, 10mM CaCl2), remember to add CaCl2 after dialysis is complete.
Q7: How many purifications can be performed using the same affinity column?
A7: This varies with the stability of the immobilized protein and the type of elution buffer used. Columns can typically be reused at least 10 times without significant loss of activity.
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