How to purify total protein from protein lysis

The aqueous solution of dilute salt and buffer system has good stability and high solubility to protein, and is the most commonly used solvent for protein extraction, usually 1-5 times the volume of raw materials, and uniform stirring is required during extraction to facilitate the dissolution of protein. The temperature of extraction depends on the nature of the active ingredient. On the one hand, the solubility of most proteins increases with increasing temperature, so a high temperature is conducive to dissolution and shortens the extraction time.

On the other hand, the increase in temperature denatures and inactivates proteins, so it is common to use low temperatures (below 5 degrees Celsius) to extract proteins and enzymes based on this consideration. In order to avoid degradation during protein extraction, proteolytic enzyme inhibitors (e.g. diisopropyl fluorophosphate, iodoacetic acid, etc.) can be added. Stool loss.

The selection of pH and salt concentration of the extract is discussed below.

1. pH value.

Protein, enzyme jujube orange god is an amphoteric electrolyte with an isoelectric point, and the pH value of the extract should be selected at the pH on both sides of the isoelectric point

range. When extracting with dilute acid or dilute alkali, it should be prevented from changing the dissociable group of the protein caused by excessive acid or alkaline, which will lead to irreversible changes in the conformation of the protein.

2. Salt concentration.

The dilute concentration promotes the dissolution of proteins, which is called salt solubilization. At the same time, the dilute salt solution has the advantage of protecting the protein from denaturation due to the partial combination of salt ions and proteins, so a small amount of neutral salts such as NaCl is added to the extract, generally in moles. liter concentration is appropriate.

Phosphate and carbonate isotonic salt solutions are often used in buffers.

1.Pre-treatment.

Proteins are released from their original tissue or dissolved state, maintaining their original state without loss.

Loss of biological activity. Commonly used methods: homogenizer crushing, hypergeneration crushing, cellulase treatment and lysozyme.

Ultrasonic disruption method: When the sound wave reaches a certain frequency, the liquid produces a cavitation effect to break the cells. The rapid vibrations caused by ultrasonic waves cause a local low air pressure in the liquid, which converts the liquid into a gas.

That is, a lot of small bubbles are formed. Due to the local pressure transition, the pressure rises again and the bubble collapses. The collapsing bubble creates a vibrating wave that is delivered into the liquid, creating a shear force that breaks the cell.

2 Coarse grading.

Separation can be done by salting out, isoelectric precipitation and organic solvent fractionation. These methods are characterized by simplicity, high throughput, and 3 subdivisions.

Further purification of the sample. After crude separation, the sample is generally small in size, and most of the miscellaneous proteins have been removed. For further purification, chromatography methods are commonly used, including gel filtration, ion exchange chromatography, adsorption chromatography, and affinity chromatography.

If necessary, electrophoresis, isoelectric focusing, etc., can also be selected as the final purification step.

Crystallization is the final step.

There is a kit in the United States, the total protein extraction kit SD001 by centrifuge column method, which only needs to be centrifuge the sample into a centrifuge tube for 30 seconds to obtain all the total protein, and the operation is extremely simple.

PBS is a phosphate buffer. In order to prevent protein denaturation due to too drastic changes in pH value.

Generally, proteins can be dissolved in an aqueous solution. I don't know what kind of protein you make? If it is a denatured protein, it may be insoluble. Different pH of PBS should be different for the optimal pH in different extraction steps.

When I say water-soluble proteins, I mean proteins in general. The surface of the human body is keratin, which is insoluble in water.

I strongly suspect that you have not learned the basic principles of biological chemistry.

If it is an intracellular soluble protein, it is necessary to use cytolysis (such as ultrasonic lysis) to release the contents, and then use column chromatography and other steps to extract; If it is an intracellular inclusion body, after the cell is broken, centrifugation, collection of precipitate, and the denaturation refolding method is used to obtain a protein with high purity;

If it is an extracellular protein, it needs to be enriched and concentrated, and then extracted by conventional methods.

1.Organic solvents such as acetone can be added to precipitate the protein, and then centrifugal collection of the precipitate, the precipitate is redissolved with deionized water, and the amount of deionized water can be added according to actual needs, so as to change the protein concentration. However, this method may impair protein activity.

2.Using a dialysis tubing with polyethylene glycol dry powder on the outside to allow the water to slowly exud, this method can maintain protein activity, but for a longer time.

3.Use rotary evaporation, but for proteins that are not temperature sensitive.

4.The lyophilizer freeze-dries, but somewhat extreme.

There are many methods such as film cassette concentration, ethanol precipitation and reconstitution.

Freeze drying, centrifugation, protein precipitation such as isometric precipitation, and ultrafiltration.

Semi-permeable membrane filtration can be adopted.

Filtration and re-dissolution of protein salting-out?

PBS or other phosphate extracts can be found in this molecular cloning guide.

The preparation method of phosphate buffered saline containing Tween 20 (referred to as PBS T) is: weigh potassium phosphate monobasic (KH2PO4), sodium phosphate dibasic (Na2HPO4·12H2O), sodium chloride (NaCl), potassium chloride (KCL), Tween 20, and add water. pbs:

Phosphate Buffered Saline PBS 1L Formula: Potassium Phosphate Monobasic (KH2PO4): , Sodium Phosphate Dibasic (Na2HPO4):

Sodium chloride (NaCl): 8g, potassium chloride (KCL), add about 800ml of deionized water to stir and dissolve thoroughly, then add concentrated hydrochloric acid to adjust pH, and finally set the volume to 1L. Store at room temperature after autoclaving.

PBS buffer (NaCl 137mmol L, KCl, Na2HPO4 10mmol L, KH2PO4 2mmol L PBS buffer is generally used as a solvent to dissolve the protective reagent, and the specific reagent generally has different proportions and formulations, which has a better effect in terms of targeting. 1x PBS buffer is the PBS configuration used and can be used directly, 2x PBS is 2x the concentration and is used with a double dilution.

PBS is generally not used to prepare buffers and is used for other purposes.

Shanghai Biotech's one-step phytoactive protein extraction kit BSP004 can be used

It's called sample buffer, and you can understand it.