immunoprecipitation mass spectrometry

Summary

Immunoprecipitation is a technique used to enrich or purify specific proteins from complex mixtures, which can then be combined with mass spectrometry to identify the interacting proteins of specific proteins.

Appliance

Detecting protein interactomics using the powerful protein characterization capabilities of mass spectrometry.

Operation method

Immunoprecipitation mass spectrometry

Materials and Instruments

Cell lysate
pH 7.4 sterile PBS, specific antibodies
Protein A/G
Sampling/sample buffer for protein blot analysis
Acetonitrile, ammonium bicarbonate, purified water, DTT, iodoacetamide

Move

For suspended cells:

1, Gently transfer the cells to be lysed to a pre-cooled microcentrifuge tube and wash the cells once with pre-cooled PBS.

2, Dispose of PBS by centrifugation and add pre-cooled lysis buffer (1 ml for every ¹⁰⁷ cells).

3, Mix at a constant rate for 5-20 min at 4 °C to allow sufficient lysis.

4, Centrifuge at 14000 g for 10 min at 4 °C, aspirate the supernatant into a clean tube on ice and discard the sediment.

For adherent cells:

1, Discard the medium and wash the cells once with ice-cooled PBS. 2, Remove PBS and add pre-cooled PBS.

2, Remove PBS, add pre-cooled cell lysate and incubate on ice for 5 min.

3, Scrape the cells from the petri dish and transfer to an EP tube.

4, Centrifuge at 14000 g at 4 °C for 10 min and transfer the supernatant to a new EP tube.

5, Pre-wash lysate: Add 50 μL of non-target antibody from the same species and isoform to 1 mL of lysate as immunoprecipitating antibody and incubate on ice for 1 h. 6, 4 °C, 5000 g, 4 °F.

6, Centrifuge at 4 °C, 5000 g for 10 min and discard the microbead precipitate, save the supernatant for immunoprecipitation.

7, Antibody incubation: Add approximately 70-100 µL of Protein A or G to a microcentrifuge tube and add 10 µL of primary antibody (based on antibody concentration).

8, Gently mix the antibody-bead mixture by turning the mixer over and incubate for 1-4 hours at 4 °C. 9, Gently mix the antibody-bead mixture by turning the mixer over and incubating for 1000 minutes at 4 °C.

9, Centrifuge at 1000-3000 g for 2 minutes at 4 °C and discard the supernatant.

10, Wash: Resuspend the beads by adding 1 mL of Lysis Buffer and centrifuge at 3000 g for 2 minutes at 4 °C. Repeat this wash step twice. Repeat this washing step twice.

11, Add 40 µL of Protein Sampling Buffer and heat at 95 °C for 5 min to elute the sample.

12, SDS-PAGE electrophoresis run: select the appropriate concentration of gel for electrophoretic separation.

13, Cut off the gel block including bromophenol blue, wash it three times with purified water, cut the gel block into 1 mm × 1 mm pellets with a small blade, and put them into clean EP tubes, the pellets can be stored at -80 °C for several months.

Sample Preparation for Mass Spectrometry

1, Colloid decolorization: Wash the colloid once with 1 ml of pure water and remove the supernatant with a pipette gun. Add 1 ml of 25 mM ammonium bicarbonate solution in 50% acetonitrile and shake for 5 min at room temperature to remove the supernatant.

2, Dehydration: Add 1 ml of pure acetonitrile and blow the pellet, the pellet will turn white immediately, incubate at room temperature for 10 min, remove the acetonitrile, and then place it in a vacuum desiccator at 37 °C for 10 min.

3, Reduction: Add 1 ml of 25 mM ammonium bicarbonate 50 mM DTT solution, incubate at 56 °C for 1 h.

4, Alkylation: After DTT reduction, cool to room temperature, quickly add 1 ml of 20 mM iodoacetamide solution prepared with 25 mM ammonium bicarbonate and leave for 45 min in a drawer protected from light.

5, Repeat steps 1 and 2 to wash the pellet for trypsin digestion.

6, Trypsin digestion: Prepare 0.5 ug of trypsin with 200 μl of 25 mM ammonium bicarbonate, add it to the dried pellet, and leave it on ice for 10 min. After the pellet absorbs the enzyme solution, place it at 37 °C and shake for about 4-10 h. 7, Aspirate the supernatant of the pellet, and then transfer the supernatant into the gelatinized gelatinized pellet.

7, Aspirate the supernatant from the pellet into a new EP tube and wash the pellet twice with 200 μl of 25 mM ammonium bicarbonate solution in 50% acetonitrile, combine the pellets into the EP tube, and place the pellet in a vacuum desiccator until it is dry, and the dried samples can be stored at -80 °C for several years.

Note: Immunoprecipitation experiments can also be performed by magnetic separation using magnetic beads.

Caveat

a. After cell lysis is complete, pre-washing of the protein lysis products is required.b. Protein A/G encapsulation of the microbeads is selected based on the species and serotype of origin of the IP antibody.c. Enrichment of specific labeled proteins can also be performed using labeled antibody-coupled microbeads.d. Sample preparation for mass spectrometry must be complete with reduction and alkylation.e. Low adsorption EP tubes are recommended for experiments.f. Avoid contact with plastics as much as possible and avoid contamination during experiments.

Common Problems

A. High background: 20,000 g centrifugation to remove insoluble material from the lysate and microbead pretreatment of the lysate.

B. Few proteins identified by mass spectrometry: add fresh protease inhibitor to increase the protein concentration of the sample. Quantify the eluate BCA, the protein concentration should be greater than 0.1 μg/μL.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/