5 Buffers & Reagents
5.1 Buffers for general assay procedures
5.2 Reagents
The following reagents are needed
| PRODUCT DESCRIPTION | Manufacturer | Part No. | Pack size |
|---|---|---|---|
| PBS tablets | Sigma | P4417-100TAB | 100 tablets |
| Protease inhibitor (for DBS samples only) | Roche | 11697498001 | 20 tabs |
| BSA | Sigma | A4503-100G | 100 g |
| EDC | Sigma | 11851335 | 5 g |
| NHS | Sigma | 10391314 | 500 mg |
| MES | Sigma | M3671 | 50 g |
| Sodium phosphate monobasic | Sigma | S8282 | 500 g |
| Tween 20 | Sigma | P1379-100ML | 100 ml |
| NaOH (sodium hydroxide) | Sigma | S5881-500G | 500 g |
5.2.1 Deionised water (dH2O)
Filter 50 mL of deionised water through a 0.2 µm filter into a sterile container.
5.2.2 PBS
1 tablet (Sigma; P4417) per 200 mL deionised water.
Filter through 0.2 µm filter into one or more sterile containers.
5.2.3 PBST (wash buffer)
PBS + 0.5 mL/L (0.05% v/v) Tween 20
Make fresh every week.
5.2.4 PBS-TBN (blocking/storage/assay buffer)
PBST + 10 mg/mL BSA + 0.5 mg/mL sodium azide
Per 100 mL PBST, add:
- 1 g BSA
- 500 µL of 10% (100 mg/mL) sodium azide solution in deionised water
- *CAUTION* - use appropriate PPE and safety measures when handling solid and liquids with sodium azide.
Filter through 0.2 µm filter and store in fridge until use.
5.3 Buffers for bead coupling
5.3.1 Activation buffer
0.1 M sodium phosphate monobasic, pH 6.2
- 1.19 g NaH2PO4 (Sigma, S8282)
- 100 mL dH2O
- Adjust pH to 6.2 with 5 or 10 M NaOH. Filter through 0.2 µm filter and store in the fridge or freeze aliquots until use.
5.3.2 Coupling buffer
50 mM MES, pH 5.0
- 0.976 g MES (Sigma, M3671)
- 100 mL dH2O
- Adjust pH to 5 with 5 or 10 M NaOH. Filter through 0.2 µm filter and store in the fridge or freeze aliquots until use.