Table 1.
Table showing key resources used in this chapter. Products or applications are identified by their manufacturers and catalog numbers or website links. Proteins and peptides that were prepared in-house are accompanied by relevant references. X = 6-aminohexanoic acid. FITC = fluorescein isothiocyanate. Dap = 2,3-diaminopropionic acid. Sox-STE7 was synthesized by alkylation of Sox-Br (2-bromomethyl-8-tertbutyldiphenylsilyloxy-5-(N,N-dimethyl) sulfonamide quinolone) to the cysteine residue of the peptide.
| Reagent or Resource | Source | Identifier and/or Notes |
|---|---|---|
| Peptides and recombinant proteins | ||
| Inactive, tagless ERK2 | Kaoud, Devkota et al. (2011); Yan et al. (2011) | Note: All proteins used in this protocol were prepared in-house. Proteins may be purchased commercially as an alternative option. |
| Active, tagless ERK2 | Waas and Dalby (2002); Yan et al. (2011) | |
| ERK2-C159S mutant | Kaoud, Devkota et al. (2011) | |
| MKK1G7B (constitutively active MKK1 mutant) | Waas and Dalby (2002) | |
| FITC-X-Lig-D (FITC-X-FQRKTLQRRNLKGLNLNL) | Lee et al. (2011); Kaoud, Mitra et al. (2011) | Note: Peptides for the anisotropy assay were prepared and labeled in-house. |
| Lig-D(Dap) (FQRKTLQRRNLK(Dap)LNLNL) | Lee et al. (2011) | |
| Sox-STE7 (FQRKTLQRRNLKGLN-LNL-XXX-TGPLSP-C(Sox)-PF) | Zamora-Olivares et al. (2014) | Note: Unlabeled peptide was purchased from Anaspec (#1570512) and labeled in-house. |
| Antibodies | ||
| Antiphospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10) mouse mAb | Cell Signaling Technology | 9106 |
| Anti-p44/42 MAPK (Erk1/2) (137F5) rabbit mAb | Cell Signaling Technology | 4695 |
| IRDye 800CW Goat (polyclonal) anti-rabbit IgG | LI-COR | 926-32211 |
| IRDye 680RD Goat (polyclonal) anti-mouse IgG | LI-COR | 926-68070 |
| Chemicals | ||
| HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) | Sigma-Aldrich | H3375 |
| KCl (potassium chloride) | Sigma-Aldrich | P9541 |
| EDTA (ethylenediaminetetraacetic acid disodium salt dihydrate) | Sigma-Aldrich | E5134 |
| EGTA (ethylene glycol-bis (β-aminoethylether)-N,N,N,N-tetraacetic acid) | Sigma-Aldrich | E3889 |
| Glycerol | Fisher Scientific | BP1605 |
| KOH (potassium hydroxide) | RICCA Chemical | 6252.90 32 |
| BSA (bovine serum albumin) | Fisher Scientific | BP1605 |
| Triton X-100 | Sigma-Aldrich | X100PC |
| DMSO (dimethyl sulfoxide) | Sigma-Aldrich | 276855 |
| MgCl2 (magnesium chloride) | Millipore Sigma | M1028 |
| ATP (adenosine 5′-triphosphate disodium salt trihydrate) | Roche | 10519987001 |
| DTT (dithiothreitol) | GoldBio | DTT100 |
| Odyssey Blocking Buffer | LI-COR | 927-40000 |
| Tris base | Sigma-Aldrich | T6066 |
| Glycine | Sigma-Aldrich | G7126 |
| NaCl | Sigma-Aldrich | S3014 |
| Tween 20 | Sigma-Aldrich | P9416 |
| Methanol | Thermo Scientific | 423950040 |
| Blotting-grade blocker (non-fat dry milk) | Bio-Rad | 1706404 |
| Key instruments | ||
| Synergy H4 multimode microplate reader (or updated version) | Agilent | Note: Recommended accessories include BioStack and barcode reader |
| Janus Automated Workstation | PerkinElmer | Note: Use with 384 MDT head |
| Odyssey Sa imaging system | LI-COR | Note: Use with Image Studio software |
| Software | ||
| GraphPad Prism (version 7 or higher) | GraphPad by Dotmatics | https://www.graphpad.com/ |
| LI-COR Image Studio | LI-COR Biosciences | https://www.licor.com/bio/image-studio/ |
| Useful websites | ||
| SciFindern | Chemical Abstracts Service (American Chemical Society) | https://scifinder-n.cas.org/ |
| Pub Chem | National Institutes of Health | https://pubchem.ncbi.nlm.nih.gov/ |