Cell Adhesion Sample Preparation
1. Remove the culture medium and wash the adherent cells twice with PBS.
2. Collect the cells into a 1.5 mL Eppendorf (EP) tube, add Lysis/Wash Buffer in proportion, and simultaneously include appropriate inhibitors such as PMSF. Mix thoroughly and incubate on ice for 5-20 minutes, stirring occasionally.
3. Centrifuge at 4°C, 12000-16000 g for 10 minutes to collect the supernatant. Place it on ice for subsequent experiments or store it long-term at -80°C.
Suspension Cell Sample Preparation
1. Centrifuge cells at 4°C, 500-1000 g for 10 minutes, collect the cells, and discard the supernatant.
2. Wash cells once with PBS, resuspending the cell pellet in PBS, and centrifuge at 4°C, 500-1000 g for 10 minutes. Collect the cells and discard the supernatant.
3. Resuspend cells in pre-cooled IP Lysis/Wash Buffer. Use 5-10 μL IP Lysis/Wash Buffer per 1 mg of cells. Add appropriate inhibitors, such as PMSF, mix thoroughly, and incubate on ice for 5-20 minutes, stirring occasionally.
4. Centrifuge at 4°C, 12000-16000 g for 10 minutes to collect the supernatant. Place it on ice for subsequent experiments or store it long-term at -80°C.
Preparation of Immunocomplexes
Note: The required amount of sample and incubation time depends on the specific antibody-antigen system, and optimization may be necessary to achieve maximum yield. The following experimental protocol is designed for the affinity purification of 5-15 μg of antibody, and adjustments can be made proportionally as needed.
1. In a centrifuge tube, combine the cell lysate of each sample with the immunoprecipitation antibody.
2. Dilute the antibody and prepared samples with IP Lysis/Wash Buffer to a final volume of 500μL-800μL.
3. Incubate at 4 ℃ for 2-4 hours or overnight to allow the formation of immunocomplexes.
Immunoprecipitation
Note: To ensure even distribution of magnetic beads, invert or gently vortex the bottle containing the beads before use.
1. Add 50-200µL of Protein A/G Magnetic Beads to a 1.5 mL centrifuge tube.
2. Add 500-1000µL of pre-chilled PBS to the beads, and gently mix.
Place the centrifuge tube in a magnetic stand to collect the beads on one side of the tube. Discard the supernatant.
3. Add 500-1000µL of IP Lysis/Wash Buffer to the tube. Invert the tube several times or gently vortex for 1 minute. Collect the beads using the magnetic stand. Discard the supernatant.
4. Add the antigen/antibody mixture to the tube containing the beads, and gently mix. Incubate at 4 ℃ for 2-4 hours or overnight.
5. Collect the beads using the magnetic stand, remove unbound samples, and store for analysis.
Add 500-1000µL of IP Lysis/Wash Buffer to the tube, gently mix, collect the beads, and discard the supernatant. Repeat the washing procedure twice.
Mass Spectrometry Sample Preparation
(I) Protein Qualification in Solution (Proceed to Step 7)
1. Elute the protein from the magnetic beads.
2. Measure the protein concentration ideally, then send the nitrogen-frozen protein solution on dry ice (specify the buffer composition and concentration); alternatively, precipitate the protein using the TCA or acetone method, air-dry, and send on dry ice or ice packs.
Sample Requirements
A. The buffer should not contain detergents such as NP40, Triton X-100, etc. B. It should be free from substances like SDS. C. Competitive elution is recommended (e.g., flag-tagged proteins eluted by competition with flag peptides).
(II) Gel Strip Identification Sample Requirements (Proceed to Step 7)
1. Add 80-100 µL of SDS-PAGE Sample Loading Buffer (1×) to a centrifuge tube, place the sample in a 100°C water bath or metal bath, and heat for 10 minutes. Separate the magnetic beads using a magnetic stand, retaining the supernatant containing the target antigen.
2. After electrophoresis, stain with Coomassie Brilliant Blue. Cut the protein bands with a clean blade, sectioning them into 1 mm^3 pieces, and promptly transfer them into labeled EP tubes for sample submission. Maintain a low temperature during sample transport by including ice packs.
Sample Requirements
A. Protein Solution: Concentration >0.1 μg/μL, total protein amount >5 μg. B. The buffer should not contain detergents such as NP40, Triton X-100, etc. C. SDS-PAGE Bands: Clearly visible bands after staining (compatible with mass spectrometry) using Coomassie or silver stain.
Notes
Proteins on PVDF or NC membranes are not suitable for mass spectrometry analysis.
For additional questions related to sample preparation, refer to the "Sample Preparation Q&A" .
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