Protein extraction experiments

Summary

Protein extraction experiments are basic operations for performing experimental analysis of proteins and can be used for (1) protein isolation (2) protein functional structure detection (3) basic operations before protein expression.

Operation method

ultrasonic crushing

Principle

After the proteins are expressed in the bacteria, the cells are broken by repeated freezing and thawing, and the proteins are precipitated by ammonium sulfate, a step that removes most of the nucleic acids, polysaccharides, lipids, and other debris.

Materials and Instruments

Transformed strain pQG11 M15 [pREP] single colony
LB amp kan liquid medium IPTG Na3PO4-12H2O NaCl EDTA Triton X-100 liquid nitrogen
Constant temperature shock incubator High-speed freezing centrifuge Centrifuge tube Ice cylinder

Move

1. Pick a single colony from a petri dish and incubate it in 15 mL of LB/amp/kan liquid medium, shaking at 120 rpm, overnight at 37°C.

2. Take 6 mL of the above bacterial solution and add it to 300 mL of fresh LB/amp/kan medium, incubate at 120 rpm at 37℃ until _A600 = 0.6, then add IPTG to make a concentration of 1 mM, and continue to incubate at 120 rpm at 37℃ for 4 hours with shaking.
3. Pour the liquid into a 50 mL conical tube and centrifuge for 10 min (5 000 rpm).
4. Pour off the supernatant and store the pellet with the conical tube in the refrigerator at -20℃.
5. Remove the tube containing the bacterial mass and place on crushed ice. After the bacterial mass has dissolved, use the remaining liquid for uniform suspension. Add 10 mL of lysis buffer for gentle suspension.
6. Freeze the bacterial solution in liquid nitrogen for 1 min, and then dissolve it by shaking in a 37℃ water bath. Repeat this three times, trying not to create a lot of air bubbles. Pour the contents of the centrifuge tube into a 50 mL high-speed centrifuge tube.
7. Centrifuge for 20 min (12,000 rpm, 4°C) and remove a total of _____ mL of supernatant, referred to as the Coarse Extract (XT); reserve 100 μL for later analysis. Thereafter, the samples should be placed on crushed ice to allow for low temperature experiments.
8. Add 5 mL of buffer A-0 to the supernatant and mix well. Pour into a beaker, place on crushed ice and allow to cool. Stir from time to time and slowly add solid ammonium sulfate _____ gm to 70% saturation, stirring for 10 minutes after addition.
9. After centrifugation for 20 min (12,000 rpm, 4°C), carefully remove the supernatant and remove the white precipitate.
10. Add 2 mL of buffer A-150 to the precipitate, and then remove the insoluble material by tabletop centrifugation (10,000 rpm, 5 min), resulting in a total of ______ mL of total protein (TP); reserve 100 uL of TP, along with the above XT, and place it under refrigeration at 4℃.
11. The rest of the solution is prepared for colloidal filtration chromatography, labeled clearly and refrigerated at 4℃.

Caveat

1. always operate in small volumes and quantities to avoid contamination and move quickly.

2. proteases are prevalent and are inhibited early and often during the steps of protein extraction.

3. dispense protein solutions, store at different temperatures and in different buffers, measure protein activity, and find optimal storage conditions.

4. refrigerate and add stabilizers. These stabilizers generally maintain the biological activity of the protein adequately.

5. Avoid repeated freezing and thawing of the enzyme. If the enzyme needs to be frozen, divide it into small tubes and freeze them.

Common Problems

1、Instruments

and appliances

Constant temperature shock incubator at 37℃; high-speed freezing centrifuge and centrifuge tubes (using 20,000 rpm centrifuge) Using high-speed centrifuge should pay attention to: centrifuge and centrifuge temperature should be pre-cooled completely, the two centrifuge tubes should be balanced in the opposite position, and centrifugal rotational speed of the centrifuge should never exceed the maximum speed limit; liquid nitrogen and ice cylinder; 37℃ warm water bath.

2, drugs and reagents

Transformation strain pQG11/M15 [pREP] single colony; LB/amp/kan and IPTG (1 M stock); Lysis buffer (50 mM _Na3PO4-12H2O, pH 7.0; 0.1 M NaCl; 0.1 mM EDTA; 0.2% Triton X-100) added before use.

Buffer A-0 (50 mM _Na3PO4, pH 7.0) Add 70 μL β-mercaptoethanol per 1 L before use to make 10 mM final concentration. Buffer A-150: Buffer A-0 is supplemented with 0.15 M NaCl; ammonium sulfate (to be dried and ground with a port).

3. Protein storage:

(1) Protein samples were snap-frozen in liquid nitrogen, taken out and added with 10-20% glycerol for long-term storage.

(2) Keep the protein samples on ice throughout the experiment.

(3) Use fresh tips when aspirating different proteins from the parent tube.

(4) Do not pour unused solution back into the parent tube.

(5) Wear gloves to avoid cross-contamination of proteins or contamination by proteases.

(6) Avoid violent shaking to avoid mixing protein denaturant in the solution.

(7) Avoid dust, which contains 60% of the sarcosine, during the experiment.

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