["Collaborating Experts | Qingqiang Lei, M.S.", "Immunology Sichuan University"], ["Reviewed by | Dr. Li-Min Ji Yang", "Polymer Chemistry and Physics Institute of Chemistry, Chinese Academy of Sciences"]
Summary
Oligomerization refers to the formation of dimers, trimers, tetramers or long chain molecules (oligomers) between monomeric compounds. In a biological context, oligomerization usually refers to the process by which biological macromolecules (e.g., proteins) form macromolecular complexes through non-covalent interactions. Oligomerization occurs in organisms in physiological or pathological states.
Principle
Protein blotting is a commonly used experimental method in molecular biology, biochemistry and immunogenetics. The basic principle is the coloration of gel electrophoresis-treated cell or biological tissue samples by specific antibodies. Information on the expression of specific proteins in the cells or tissues analyzed is obtained by analyzing the location and depth of the staining.
Appliance
Immunoprecipitation is primarily a technique used to study protein-protein interactions, and then protein blotting can be used to detect protein oligomerization.
Operation method
Protein blotting to detect protein oligomerization
Principle
Protein blotting is a commonly used experimental method in molecular biology, biochemistry and immunogenetics. The basic principle is the coloration of gel electrophoresis-treated cell or biological tissue samples by specific antibodies. Information on the expression of specific proteins in the cells or tissues analyzed is obtained by analyzing the location and depth of the staining.
Materials and Instruments
Materials:
6-well plate, pipette gun, 1.5 mL EP tube
Ultrasonic cell breaker, electrophoretic membrane transfer device PBS
Protein lysate, protease inhibitor
SDS Sampling Buffer
TBST electrophoresis buffer: weigh 10 g of SDS, 30.25 g of Glycine, 144.25 g of Tris-base, and mix with ddH
2
O to 10 L
Membrane transfer buffer: weigh 144 g of Glycine, 30.2 g of Tris-base, and dilute with ddH
2
O to 10 L
Move
1. Inoculate the cells required for the experiment (usually 1×106 cells) into a 10 cm cell culture dish and transfect the target plasmid into the cells by calcium phosphate transfection after about 12 h. Collect the cells after 18-24 h. Wash the cells with 1-2 mL of PBS/dish at 4 °C, centrifuge at 3000 rpm for 3 mins, and aspirate off the PBS. The cells were washed with 1-2 mL of PBS/dish at 4 °C, centrifuged at 3000 rpm for 3 mins, and the PBS was aspirated off. 2.
2. Add the appropriate volume of protease inhibitor to the protein lysate, 1 mL per dish, and place on ice or in the refrigerator to lysate for 10-15 min. Collect the lysate into a 1.5 mL EP tube, taking care to avoid foaming.
3. Transfer the lysate to the ultrasonic cell breaker, set the power at 10% (the actual power is 96 w), sonicate for 3 s, pause for 7 s, and perform 5 times. When the sample is thick, freeze and thaw in liquid nitrogen 3 times after sonication. Centrifuge the sonicated sample at 4 °C for 10 min at 12000 rpm. 4.
4. After centrifugation, the supernatant is divided into three fractions, 50 mL for the lysate and 450 mL for the experimental group. 50 mL of the lysate is added directly to 10 μL of 6× SDS sample buffer and heated for 10 minutes at 95 °C. 5.
5. Add 1.5 mL of the corresponding antibody to the control group and rotate in a rotary shaker at 4 °C for 2h~4h (depending on the nature of the protein), or overnight if the signal is weak. 6. Add 35 mL of protein G to the control group.
6. Add 35 mL of protein G and spin for 1 h at 4 °C on a rotary shaker.
7. Utilize prelysis buffer containing 0.5 M NaCl as wash buffer, where NaCl is added fresh. After removing the sample from the rotary shaker, centrifuge the sample at 10,000 rpm for 30 s. Aspirate the supernatant with a vacuum pump, add 1 mL of wash buffer to the precipitate, mix upside down, centrifuge, and aspirate the supernatant, and repeat this procedure three to four times. Use a microsampler to aspirate the remaining wash buffer in the EP tube, add 50 mL of 1× SDS sample buffer, centrifuge instantly to ensure that the protein G at the bottom of the tube is fully infiltrated in the sample buffer, and heat denaturation for 10 min at 95 °C on a dry thermostat. 8.
8. After that, use about 500 mL of 1× above electrophoresis buffer for each electrophoresis tank. After energizing the electrophoresis tank for 15-20 min at 70 V, adjust the voltage to 130 V, and then turn off the electrophoresis apparatus and remove the plates after 90-120 min. 9. transfer the membrane.
9. Transfer the membrane, configure 1× of the above transfer solution, activate the PVDF membrane in methanol for 10-15 s and then soak it in the transfer solution, and soak the removed adhesive plate in the transfer solution for 3-5 min. After attaching the filter paper, adhesive, and membrane sequentially, use 700 mL of the above transfer solution in each transfer tank, and transfer the membrane at 200 mA for 120 min. 10.
10. Close the transferred PVDF membrane with 5% skimmed milk for 1 h at room temperature, wash off the milk and apply the corresponding primary antibody. 11.
11. Generally, after incubation at room temperature for 1 h or overnight at 4 °C (endogenous antibodies with weak signals are usually incubated overnight at 4 °C), wash the membrane with TBS containing 0.1% Tween-20 three to four times, each time for 10-15 min (generally depending on the nature of the antibody); then add the corresponding secondary antibody attached with horseradish peroxidase (HRP) (configured with 2% skimmed milk), incubate at room temperature for 1-4 h or 4 °C, and then incubate at room temperature for 1-4 h or 4 °C, and then incubate at room temperature for 1-4 h with 5% skimmed milk. Incubate at room temperature for 1-4 h or in a refrigerator at 4 °C for 8 h (depending on the nature of the protein). 12. Then dissolve the antibody in TBST.
12. The membrane is washed again with TBST solution as above. Detect the target bands using ECL chemiluminescent detection solutions of different intensities. 13.
13. Determine if the protein is oligomerized based on the molecular weight of the blot. If there is a band at 4 times the molecular weight, set up various controls to determine if that is the tetramerized form of the target protein.
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