2.2 LRC-TriCEPS
Methodology for
Identifying Binding
Partners
This section provides details on the materials needed to perform an
LRC-TriCEPS experiment.
1. 5-methoxyanthranilic acid (Sigma-Aldrich).
2. Acetonitrile LC-MS grade (Fisher Scientific).
3. Alkyne agarose.
4. Ammonium bicarbonate.
5. Calcium chloride (CaCl 2 ).
6. Copper(II)sulfate (CuSO 4 .
7. Ethylenediaminetetraacetic acid (EDTA).
8. Formic acid, 98–100% (vol/vol).
9. Glycine.
10. HEPES pH 8.2, 1 M buffer solution.
11. Iodoacetamide (Sigma-Aldrich).
12. Isopropanol for molecular biology, !99.5%.
13. Mobicol spin columns (Life Systems Design).
14. Mobicol filter 35 μm pore size (Life Systems Design).
15. PBS, pH 7.4.
16. Phosphoric acid 85 wt.% (H 3 PO 4 ).
17. Protease inhibitors cOmplete (Sigma-Aldrich).
18. Rapigest SF (waters).
19. Sequencing grade modified trypsin (Promega).
20. Sodium ascorbate.
21. Sodium bicarbonate.
22. Sodium chloride.
23. Sodium dodecyl sulfate (SDS, Sigma-Aldrich).
24. Sodium hydroxide solution.
25. Sodium-metaperiodate (NaIO 4 , Sigma-Aldrich).
26. TriCEPS v.3.0 (Dualsystems Biotech).
27. Tris (2-carboxyethyl)phosphine hydrochloride (TCEP, SigmaAldrich).
28. Tris (3-hydroxypropyltriazolylmethyl)amine (THPTA, SigmaAldrich).
29. Tris (hydroxymethyl)aminomethane base.
30. Urea.
31. Water HPLC gradient grade.
2.2.1 Buffer Preparation
1. Ligand coupling: HEPES 25 mM pH 8.2: Dilute 1 M HEPES
with HPLC water and adjust the pH to 8.2 with NaOH.
102
Kyle S. Feldman et al.
Methodology for
Identifying Binding
Partners
This section provides details on the materials needed to perform an
LRC-TriCEPS experiment.
1. 5-methoxyanthranilic acid (Sigma-Aldrich).
2. Acetonitrile LC-MS grade (Fisher Scientific).
3. Alkyne agarose.
4. Ammonium bicarbonate.
5. Calcium chloride (CaCl 2 ).
6. Copper(II)sulfate (CuSO 4 .
7. Ethylenediaminetetraacetic acid (EDTA).
8. Formic acid, 98–100% (vol/vol).
9. Glycine.
10. HEPES pH 8.2, 1 M buffer solution.
11. Iodoacetamide (Sigma-Aldrich).
12. Isopropanol for molecular biology, !99.5%.
13. Mobicol spin columns (Life Systems Design).
14. Mobicol filter 35 μm pore size (Life Systems Design).
15. PBS, pH 7.4.
16. Phosphoric acid 85 wt.% (H 3 PO 4 ).
17. Protease inhibitors cOmplete (Sigma-Aldrich).
18. Rapigest SF (waters).
19. Sequencing grade modified trypsin (Promega).
20. Sodium ascorbate.
21. Sodium bicarbonate.
22. Sodium chloride.
23. Sodium dodecyl sulfate (SDS, Sigma-Aldrich).
24. Sodium hydroxide solution.
25. Sodium-metaperiodate (NaIO 4 , Sigma-Aldrich).
26. TriCEPS v.3.0 (Dualsystems Biotech).
27. Tris (2-carboxyethyl)phosphine hydrochloride (TCEP, SigmaAldrich).
28. Tris (3-hydroxypropyltriazolylmethyl)amine (THPTA, SigmaAldrich).
29. Tris (hydroxymethyl)aminomethane base.
30. Urea.
31. Water HPLC gradient grade.
2.2.1 Buffer Preparation
1. Ligand coupling: HEPES 25 mM pH 8.2: Dilute 1 M HEPES
with HPLC water and adjust the pH to 8.2 with NaOH.
102
Kyle S. Feldman et al.