Preparation of antiserum and detection of proteins by Western blotting using the starlet sea anemone, Nematostella vectensis
Among basal animals, the starlet sea anemone Nematostella vectensis (phylum Cnidaria) has emerged as a leading laboratory model organism. Herein, we describe techniques to prepare antiserum against Nematostella proteins and to detect Nematostella proteins by Western blotting of extracts from animals or tissue culture cells overexpressing Nematostella cDNAs. These protocols may also be used to complement a variety of cellular and molecular techniques being used by the increasing number of investigators who are focusing on gene regulatory systems of basal animals, such as sponges, corals, and placozoans.
Abbreviations: aa, amino acid(s); ASW, artificial sea water; BSA, bovine serum albumin; EST, expressed sequence tag; HRP, horseradish peroxidase; KLH, Keyhole Limpet hemocyanin; NGS, normal goat serum; Nv, Nematostella vectensis; PBS, phosphate-buffered saline; SDS, Sodium dodecyl sulfate; TBS, Tris-buffered saline; TBST, Tris-buffered saline-Tween-20.
Figure 1
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Table 1 Antisera used for Western blotting.
Table 2 Troubleshooting table.
Posted 13 Dec, 2012
Preparation of antiserum and detection of proteins by Western blotting using the starlet sea anemone, Nematostella vectensis
Posted 13 Dec, 2012
Among basal animals, the starlet sea anemone Nematostella vectensis (phylum Cnidaria) has emerged as a leading laboratory model organism. Herein, we describe techniques to prepare antiserum against Nematostella proteins and to detect Nematostella proteins by Western blotting of extracts from animals or tissue culture cells overexpressing Nematostella cDNAs. These protocols may also be used to complement a variety of cellular and molecular techniques being used by the increasing number of investigators who are focusing on gene regulatory systems of basal animals, such as sponges, corals, and placozoans.
Abbreviations: aa, amino acid(s); ASW, artificial sea water; BSA, bovine serum albumin; EST, expressed sequence tag; HRP, horseradish peroxidase; KLH, Keyhole Limpet hemocyanin; NGS, normal goat serum; Nv, Nematostella vectensis; PBS, phosphate-buffered saline; SDS, Sodium dodecyl sulfate; TBS, Tris-buffered saline; TBST, Tris-buffered saline-Tween-20.
Figure 1
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