Western Blotting Protein Extraction Protocol

Protein extracts for Western blotting were prepared following cell fixation with trichloroacetic acid and bead beating. Extracts were then separated by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes. Antibodies used for Western detection were α-Clb5 (Santa Cruz Biotechnology, sc20170), α-Clb2 (Santa Cruz Biotechnology, sc9071), α-Sic1 (Santa Cruz Biotechnology, sc50441), α-Orc6 (clone SB49), α-Orc2 [a gift from S. P. Bell (71 (link))], α-Rga2 and α-Boi1 [a gift from D. McCusker (51 (link))], α-Cdc24 [a gift from M. Peter (72 (link))], α-myc (clone 9E10), α–hemagglutinin (HA) (clone 12CA5), α-Pk (Bio-Rad, clone SV5-Pk1; Abcam, ab15828), and α-tubulin (Crick cell services, clone TAT-1).

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Pirincci Ercan D., Chrétien F., Chakravarty P., Flynn H.R., Snijders A.P, & Uhlmann F. (2021). Budding yeast relies on G1 cyclin specificity to couple cell cycle progression with morphogenetic development. Science Advances, 7(23), eabg0007.

Publication 2021

α-Clb5 α-Clb2 α-Sic1 α-Orc6 α-Orc2 SV5-Pk1

Antibodies Cell Clone Hemagglutinin Membranes Nitrocellulose Orc2 Orc6 Protein Sds polyacrylamide gel electrophoresis Trichloroacetic acid α tubulin

Corresponding Organization : The Francis Crick Institute

Top 5 similar protocols

Variable analysis

independent variables

Cell fixation with trichloroacetic acid
Bead beating

dependent variables

Protein expression levels of Clb5, Clb2, Sic1, Orc6, Orc2, Rga2, Boi1, Cdc24

control variables

SDS–polyacrylamide gel electrophoresis (PAGE)
Transfer to nitrocellulose membranes
Primary antibodies used for Western detection

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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