Protein Detection in Zebrafish Cells

Zebrafish embryos were dechorionated and deyolked, and cells were collected as described [84] (link). Cell pellets or adult tissues were either directly dissolved in SDS loading buffer as described [84] (link), or first lysed in chilled CSH buffer (50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 1 mM EDTA, 1% Triton-X100, supplemented with cOmplete Protease Inhibitor Cocktail, Roche), followed by protein concentration determination. 10–12% SDS-PAGE, blotting on nitrocellulose membrane, Ponceau staining and immunodetection were carried out as described [84] (link). Used primary antibodies were: anti-Myc, 9B11 (mouse, Cell Signaling Technology; 1∶2000); anti-p63, 4A4 (mouse, Santa Cruz Technologies, against aa 1–205 of human ΔNp63), D-9 (mouse, Santa Cruz Biotechnology, against aa 15–151 of human ΔNp63), H-137 (rabbit, Santa Cruz Biotechnology, against aa 15–151 of human ΔNp63), H-129 (rabbit, Santa Cruz Biotechnology, against aa 513–641 at C-terminus of human TAp63α).

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Fischer B., Metzger M., Richardson R., Knyphausen P., Ramezani T., Franzen R., Schmelzer E., Bloch W., Carney T.J, & Hammerschmidt M. (2014). p53 and TAp63 Promote Keratinocyte Proliferation and Differentiation in Breeding Tubercles of the Zebrafish. PLoS Genetics, 10(1), e1004048.

Publication 2014

cOmplete Protease Inhibitor Cocktail anti-Myc, 9B11 H-137 H-129

Adult Antibodies Buffer Cell Edta Embryos Human Membrane Mouse Nacl Nitrocellulose Pellets Protease inhibitor Protein Rabbit Sds page Tissues Tris Triton x100 Zebrafish

Corresponding Organization : Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases

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Variable analysis

independent variables

Dechorionation and deyolking of zebrafish embryos

dependent variables

Protein expression levels measured by western blotting

control variables

Cell pellets or adult tissues were either directly dissolved in SDS loading buffer or first lysed in chilled CSH buffer (50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 1 mM EDTA, 1% Triton-X100, supplemented with cOmplete Protease Inhibitor Cocktail, Roche) followed by protein concentration determination
10-12% SDS-PAGE, blotting on nitrocellulose membrane, Ponceau staining and immunodetection were carried out in a standardized manner

controls

Positive controls: None specified
Negative controls: None specified

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