Table 1.
Reagent | Antigen/conjugate | Host | Type | Source | Catalog no. | Lot no. | Incubation | Dilution |
---|---|---|---|---|---|---|---|---|
Immunoperoxidase reagents | ||||||||
Primary | p44/42 MAP kinases a | Rb | Polyclonal IgG | CST | 9101 | 12, 13 b | 72 h, 4°C | 1:1000 |
Secondary | Anti-rabbit IgG | Gt | Biotinylated | KPL | 71-00-30 | Varies | 2 h, RT | 1 part in 3 |
Conjugate | Streptavidin | HRP conjugated | KPL | 71-00-38 | Varies | 1–1.5 h, RT | 1 part in 2 | |
Chromagen | DAB | HistoMark Enhanced Black kit | KPL | 54-75-00 | Varies | 4 min, RT | 12% (v/v) c | |
Immunofluorescence reagents | ||||||||
Primary | p44/42 MAP kinases a | Ms | Monoclonal IgG | CST | 9106 | 8 d | 72 h, 4°C | 1:1000 |
Secondary | Anti-mouse IgG e | Dk | Cy3 conjugated | Jackson | 715-165-150 | 65442 | 8–12 h, 4°C | 1:500 |
Primary | CRH | Rb | Polyclonal IgG | W. Vale | C-70 | 72 h, 4°C | 1:4000 | |
Secondary | Anti-rabbit IgG f | Dk | Biotinylated | Jackson | 711-065-152 | Varies | 6–8 h, RT | 1:500 |
Conjugate | Streptavidin | Alexa 488 conjugated | Invitrogen | S-11223 | 37350A | 8–12 h, 4°C | 1:500 |
Rb, Rabbit; Gt, goat; Ms, mouse; Dk, donkey; CST, Cell Signaling Technology (Danvers, MA); KPL, Kirkegaard & Perry Labs (Gaithersburg, MD); Jackson, Jackson ImmunoResearch (West Grove, PA). W. Vale is at The Salk Institute (La Jolla, CA). Invitrogen is located in Carlsbad, CA.
aThe specific epitope recognized is for phosphoryl modifications of human p44/42 MAP kinases at Thr202 and Tyr404 (Thr183 and Tyr185 in rat ERK2). These antibodies recognize single bands on immunoblots, corresponding to ERK1 and ERK2, respectively (according to supplier's data).
bThese lots were assayed by the supplier in February 2002 and July 2002, respectively.
cFor 50 ml of solution, 5 ml of the kit's Enhanced Buffer and 1 ml of its DAB solution were used (thus, 6 ml for every 50 ml, or 12% final concentration). The remaining reagents were as follows: 1 ml of H2O2 and 43 ml of distilled H2O.
dThis lot was assayed by the supplier in August 2004.
eThis secondary was rabbit serum adsorbed.
fThis secondary was mouse serum adsorbed.