Carboxy latex microspheres: 2.5ml, 1.5um, 20mg/ml
EDAC (1-ethy1-3-(3-dimethylaminopropy1)carbodiimide): 0.10g
15ml tip centrifuge tube: 5 tubes
High-speed centrifuge or filter membrane
0.05M MES buffer (pH 5.2): 2 X 175ml
Quenching solution (1M Glycine, pH 8.0): 25ml
Washing buffer: 125ml
1 Pipette 0.5ml (10mg) of carboxyl microspheres into a 15ml tipped centrifuge tube, centrifuge at 25000 rpm for 30 minutes, and discard the supernatant carefully with a pipette.
2 Add 5ml MES buffer and mix well. Centrifuge at 25000 rpm for 30 minutes until the supernatant becomes clear. Use a pipette to carefully discard the supernatant.
3 Repeat step 2 three times. After the last wash, resuspend the microspheres in 5ml MES buffer.
4 Take EDAC out of the refrigerated place and place it at room temperature for 30 minutes, accurately weigh the required EDAC (1.6mg EDAC/mg magnetic beads) into the centrifuge tube containing the microspheres.
5 Shake vigorously.
6 Place the centrifuge tube on the rotary mixer to activate the reaction for 30 minutes at room temperature. Be careful not to allow the microspheres to precipitate together During the reaction.
7 Centrifugation at 25000 rpm for 30 minutes, until the supernatant becomes clear, carefully discard the supernatant with a pipette.
8 Repeat step 2 four times.
1 Calculate the amount of protein/antibody to be coupled. Generally, 20-500ug protein (antibody) can be coupled per mg of activated carboxyl magnetic beads. Some carrier proteins such as BSA Fraction V can be added to increase the total protein amount in the reaction system. It can play certain blocking role to ensure the correct direction of antibody coupling.
2 Add the protein/antibody to 5ml MES buffer
3 Pipette 50ul protein/antibody solution in 950ul EMS buffer solution, make a 1:20 dilution, label it as pre-coupling protein solution, and set it aside for subsequent calculation of coupling rate.
4 Add the remaining protein solution to a centrifuge tube containing activated microspheres, shake vigorously to mix, and place the centrifuge tube on a rotating mixer at room temperature for 16-24 hours.
5 Centrifuge at high speed until the supernatant becomes clear. Use a pipette to carefully collect the supernatant and mark it as the protein solution after coupling for calculating the coupling rate
6 Resuspend the microspheres in 5ml MES buffer, shake well. Centrifuge at 25000 rpm for 30 minutes, until the supernatant becomes clear, carefully discard the supernatant with a pipette
7 Repeat step 6 once
8 Add 5ml quenching solution, shake well, place the centrifuge tube on the rotating mixer for 30 min at room temperature
9 Centrifuge at high speed until the supernatant becomes clear, then carefully remove the supernatant with a pipette
Wash and store the coupled magnetic beads
1 Add 5ml washing buffer and shake vigorously to mix. Centrifuge at high speed until the supernatant becomes clear, carefully remove the supernatant with a pipette
2 Repeat step 1 three times
3 After the last wash, resuspend the microspheres in 2ml washing buffer, at this time the concentration of the microspheres is about 5mg/ml
4 Store at 2-8°C. Freezing, drying, and centrifugation may cause irreversible accumulation of microspheres and affect the experimental results.
Note: This section provides a general procedure. Different types of protein/antibodies of different quality can be optimized on this basis to obtain the best labeling results.