氨基修饰的微球共价偶联蛋白

ipt 2017-1-22 1520

A. Reagent Preparation
1. Microspheres: Amino-Modified Polystyrene-based microspheres, 10% solids.
2. Wash Buffer: 0.1M Phosphate Buffered Saline (PBS) pH 7.4.
Make phosphate buffer by adding 0.1M NaH2PO4 to 0.1M Na2HPO4 until the pH becomes 7.4. To 200ml of the phosphate buffer above, add 8.77g of NaCl and make up the volume to one liter with deionized water. Adjust pH, if necessary, back to 7.4 with dilute HCl or NaOH.
3. Glutaraldehyde Solution: 8% Glutaraldehyde (E.M. grade) in 0.1M PBS pH 7.4.
**See note following this protocol regarding monomeric glutaraldehyde.
4. Protein Solution: 400-500g protein in 1ml of Wash Buffer. (~20-30mg protein/gram microspheres)
5. Quenching Solution: 0.15ml of ethanolamine (2-aminoethanol) in 4.8ml Wash Buffer. [This will quench any unreacted amino groups on the particle surface.]
6. Blocking Solution: 10 mg/ml BSA in Wash Buffer.
7. Storage Buffer: Wash Buffer containing 10 mg/ml BSA and 5% glycerol. (0.1% sodium azide (NaN3) may be added as a preservative if desired.)

B. Microsphere Preparation
1. Add 1ml Wash Buffer to 250l microsphere suspension (25 mg particles).
2. Centrifuge, discard supernatant, resuspend pellet in 1.5 ml Wash Buffer.
3. Repeat Step 2 twice.
4. Centrifuge, decant and discard supernatant.

C. Glutaraldehyde Activation/Washing

1. Resuspend pellet in 1ml of Glutaraldehyde Solution.
2. Incubate overnight at room temperature with gentle end-to-end mixing.
3. Centrifuge, decant and discard supernatant, resuspend in Wash Buffer.
4. Repeat Step 3.
5. Centrifuge, decant and discard supernatant.

D. Protein Coupling/Washing
1. Resuspend pellet in 1ml of Protein Solution.
2. Incubate 4-5 hours at room temperature with gentle end-to-end mixing.
3. Centrifuge, decant supernatant. Save the supernatant for protein determination.

E. Quenching
1. Resuspend the pellet in 1ml of Quenching Solution.
2. Incubate for 30 minutes at room temperature with gentle end-to-end mixing.
3. Centrifuge, decant and discard supernatant.

F. Blocking
1. Resuspend the pellet in 1ml of Blocking Solution.
2. Incubate for 30 minutes at room temperature with gentle end-to-end mixing.
3. Centrifuge, decant and discard supernatant. Resuspend the pellet in 1ml of Blocking Solution.
4. Centrifuge, decant and discard supernatant.

G. Storage
1. Resuspend the pellet in 1ml of Storage Buffer.
2. Store at 4°C.

**Purifying Glutaraldehyde (GA):
We recommend using pure monomeric glutaraldehyde for coupling proteins to amino-modified microspheres. Monomeric GA (absorbance peak 280nm) can be easily purified from contaminating polymeric GA (absorbance peak 235nm) with activated charcoal 5% (w/v) and subsequent filtration (repeat 3-4 times). To see the purification, make a UV scan (230-300nm). Subsequent storage of the purified glutaraldehyde at -20°C is recommended.10

FROM: POLYSCIENCES
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