Protein purification topic | protein purification tossed for half a month without results, watch the sharing?
At present, there are many purification methods for recombinant proteins produced by genetic engineering technology, which can be divided into precipitation technology, chromatography technology, double-liquid phase extraction technology, etc. No matter which method is used, the principle of separation and purification is to use the difference of its physical and chemical properties. Physical properties include molecular size, shape, solubility, and chemical properties include isoelectric point, hydrophobicity, and affinity with other molecules. nature, etc. The purification of protein is roughly divided into two stages: crude separation stage and fine purification. Crude separation mainly separates the target protein from other cellular components such as RNA, DNA, etc., such as ammonium sulfate precipitation. Fine purification is to distinguish the target protein from other proteins with similar size and physicochemical properties. The commonly used fine chromatography techniques are divided into two categories according to the principle: non-adsorption chromatography and adsorption chromatography. However, the following difficulties are often encountered in the protein purification process:
- The column is easy to block and the purification speed is slow;
- The protein is unstable and prone to turbidity and denaturation;
- For normally expressed proteins, the purification efficiency is very low;
- The purified protein has more heterobands.
Seeing that the summer vacation is coming to an end, I originally planned to do experiments and release myself, but the data has not come out. Others do protein purification in a few days. I can only do protein purification manually...
I had to ask the professor, how to quickly get the protein purification experiment done?
Rookie: Teacher, the nickel column of this instrument seems to be blocked. It takes a long time to start the instrument every time it is turned on, and the baseline drifts too much. The time for balancing is getting longer and longer.
Professor: The nickel column of this machine has just been replaced. If the impurity particles in your sample are handled well and the liquid-to-material ratio is also appropriate, it depends on whether the protein has flocculent precipitation when purifying the supernatant. Add DTT to reduce it as soon as possible. As for the problem that the baseline is uneven for a long time, my classmates have reported it to me many times, but the instrument has been used for too long, and I am also very helpless.
Rookie: Teacher, there will be turbidity during the experiment, what is the reason for this?
Professor: It is possible that the protein you are experimenting with is unstable. You need to check whether the buffer system is suitable and whether the temperature of the environment is too high. You can add sodium sarcosinate to quickly denature the protein, and the turbidity will be eliminated.
Rookie: Teacher, protein often does not hang on the column, what should I do?
Professor: There are many possible reasons, mainly including the following five:
- Improper ultrasonic power: too large makes the protein carbonized, and too small protein does not release. It is recommended to adjust the ultrasonic power or add lysozyme before ultrasonication to increase the cell disruption rate.
- Inappropriate buffer conditions: The concentration of metal ion chelators such as EDTA and citric acid in the buffer should not be too high, and the pH can also be appropriately increased.
- The His tag is not fully exposed: Add an appropriate amount of denaturing agent such as urea or guanidine hydrochloride to the protein for purification or change the length or position of His at the upstream molecular level to expose it on the surface of the protein.
- The His tag is not fully exposed: Add an appropriate amount of denaturing agent such as urea or guanidine hydrochloride to the protein to purify or change the length or position of His at the upstream molecular level to expose it on the surface of the protein.
- Column overload: replace a large volume column or multiple columns in series.
Rookie: The protein cannot be eluted after hanging on the column?
Professor: There are three main reasons for the inability to elute:
- The elution conditions are too mild: increase the imidazole concentration in the buffer or lower the buffer pH appropriately.
- Non-specific hydrophobic or other interactions: Add non-ionic detergent or increase NaCl concentration.
- Protein precipitation: appropriately reduce the sample amount or protein concentration, and use imidazole for linear elution; use additives or change the concentration of NaCl, or elute under denaturing conditions.
Cainiao: There are a lot of impurities after protein elution, how to deal with it?
Professor: There are three main reasons for the high protein heterobands after elution:
- Protease degradation of part of the target protein: Add protease inhibitors to improve.
- The impurity protein has high affinity with the elution column: optimize the concentration of imidazole or improve the concentration of pH and NaCl.
- Interaction between impurity protein and target protein: Add detergent or reducing agent before ultrasonication to reduce non-specific effects.
Related products are recommended as follows:
|BMR2001||PurKine™ His-Tag Ni-NTA Resin||Purify tag protein|
|KTP2001||PurKine™ His-Tag Protein Purification Kit (Ni-NTA)|
|BMR2010||PurKine™ GST-Tag Glutathione Resin|
|KTP2010||PurKine™ GST-Tag Protein Purification Kit (Glutathione)|
|BMR2020||PurKine™ MBP-Tag Dextrin Resin|
|KTP2020||PurKine™ MBP-Tag Protein Purification Kit (Dextrin)|
|BMR2030||PurKine™ Biotin-Tag Streptavidin Resin 6FF|
|KTP2030||PurKine™ Biotin-Tag Protein Purification Kit (Streptavidin)|
|BMR2040||PurKine™ Strep II-Tag Strep-Tactin Resin 4FF|
|BMR2070||PurKine™ Protein A/G Resin 4FF||Purify antibody
|KTP2070||PurKine™ Antibody Purification Kit (Protein A/G)|
|BMR2140||PurKine™ Endotoxin Removal Resin||Remove harmful substances LPS|
|KTP2140||PurKine™ Endotoxin Removal Kit (Polymyxin B)|
The technology of protein purification still has many knowledge points worthy of attention. It has been applied in a wide range of fields, plays a pivotal position in the development of the biological industry, and is continuously attracting the attention of biological scientists.
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Abbkine focuses on proteomics and cytology and is committed to the innovation and development of antibodies, proteins, analytical reagents and kits to become a key enabler of life science research and development, drug discovery and other fields. We provide you with the most popular products for the users of protein and immunology research, from the basic immunology products, such as protein extraction quantification, to internal reference labeled antibodies, primary antibodies and secondary antibodies for immunology experiments. Cell research users' favorite products, from dyes and kits for cell state detection, organelle extraction kits, cell substructure staining tracer and cell metabolism detection products, to cytokines and protein detection kits for cell culture, just to help your research career!
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