Western Blot Analysis of Trypanosoma Proteins

Western blot analysis was done as described previously [48 (link)], using a 1:1,000 dilution of polyclonal rabbit anti-PKAR3, anti-TbPKAR1 [7 (link)], anti-TbPKAC1/C2 [12 (link)] and anti-TbSAXO [25 (link)]. Rabbit antibodies against TbPKAC3 were used at 1:250 dilution [12 (link)]. For western blot detection of proteins recombinantly expressed in L. tarentolae, mouse anti-His (Bio-Rad) and mouse anti-Strep (Qiagen) were used. The secondary antibodies IRDye680LT goat anti-rabbit (1:50,000; LICOR) and IRDye800CW goat anti-mouse (1:10,000; LICOR) were used for detection with the Odyssey CLx imaging system (LICOR). Rabbit antiserum against L. donovani HSP83 [28 (link)] or β-tubulin (Cell Signalling) were used as protein loading markers.

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Fischer Weinberger R., Bachmaier S., Ober V., Githure G.B., Dandugudumula R., Phan I.Q., Almoznino M., Polatoglou E., Tsigankov P., Nitzan Koren R., Myler P.J., Boshart M, & Zilberstein D. (2024). A divergent protein kinase A regulatory subunit essential for morphogenesis of the human pathogen Leishmania. PLOS Pathogens, 20(3), e1012073.

Publication 2024

mouse anti-His mouse anti-Strep IRDye680LT goat anti-rabbit IRDye800CW goat anti-mouse Odyssey CLx imaging system β-tubulin

Corresponding Organization : University of Washington

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Variable analysis

independent variables

Dilution of polyclonal rabbit anti-PKAR3, anti-TbPKAR1, anti-TbPKAC1/C2, and anti-TbSAXO antibodies
Dilution of rabbit antibodies against TbPKAC3

dependent variables

Western blot detection of proteins recombinantly expressed in L. tarentolae

control variables

Rabbit antiserum against L. donovani HSP83 or β-tubulin (Cell Signalling) used as protein loading markers

controls

Positive control: Rabbit antiserum against L. donovani HSP83 or β-tubulin used as protein loading markers
Negative control: Not explicitly mentioned

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