Sperm Protein Expression Analysis

Basic protein extracts (equivalent to 1.5 million sperm per well) were separated on 15% AU-PAGE and transferred to PVDF membranes using the Trans-Blot Turbo System (Bio-Rad). Membranes were blocked in 1:1 TBS-0.1% Tween 20 (TBST) ChemiBLOCKER (Sigma-Aldrich/Merck) for 1 h at room temperature. Primary antibodies [ODF2 (Proteintech; 12058-1-AP; 1:500), GPX4 (Abcam; ab125066; 1:1000), H3 (Abcam; ab1791; 1:1000), H2A.L.2 (Govin et al., 2007 (link); 1:1000), SPAG8 (Proteintech; 13915-1-AP; 1:500), PRM1 (1:1000), PRM2 (1:1000)] were diluted in blocking solution and membranes were incubated at 4°C overnight. After washing in TBST, the membranes were incubated with secondary antibodies [polyclonal goat anti-rabbit IgG/HRP (Agilent Technologies/Dako; P044801-2; 1:2000), polyclonal rabbit anti-mouse IgG/HRP (Agilent Technologies/Dako; P026002-2; 1:1000)] for 1 h at room temperature. Following washing in TBST, the signals were detected using WESTAR NOVA 2.0 chemiluminescent substrate (Cyanagen) and the ChemiDoc MP Imaging system (Bio-Rad).

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Merges G.E., Meier J., Schneider S., Kruse A., Fröbius A.C., Kirfel G., Steger K., Arévalo L, & Schorle H. (2022). Loss of Prm1 leads to defective chromatin protamination, impaired PRM2 processing, reduced sperm motility and subfertility in male mice. Development (Cambridge, England), 149(12), dev200330.

Publication 2022

Trans-Blot Turbo System ChemiBLOCKER ab125066 ab1791 polyclonal goat anti-rabbit IgG/HRP WESTAR NOVA 2.0 chemiluminescent substrate ChemiDoc MP Imaging system

Anti igg Antibodies Goat Gpx4 Membranes Mouse Protein Pvdf Rabbit Sperm Tween 20

Corresponding Organization :

Other organizations : University Hospital Bonn, University of Giessen, University of Bonn

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Variable analysis

independent variables

Protein extracts (equivalent to 1.5 million sperm per well)

dependent variables

Protein expression levels of ODF2, GPX4, H3, H2A.L.2, SPAG8, PRM1, and PRM2

control variables

15% AU-PAGE gel used for protein separation
PVDF membranes used for protein transfer
Trans-Blot Turbo System (Bio-Rad) used for protein transfer
Blocking solution: 1:1 TBS-0.1% Tween 20 (TBST) ChemiBLOCKER (Sigma-Aldrich/Merck)
Blocking duration: 1 h at room temperature
Primary antibody dilutions: ODF2 (1:500), GPX4 (1:1000), H3 (1:1000), H2A.L.2 (1:1000), SPAG8 (1:500), PRM1 (1:1000), PRM2 (1:1000)
Primary antibody incubation: 4°C overnight
Secondary antibody dilutions: polyclonal goat anti-rabbit IgG/HRP (1:2000), polyclonal rabbit anti-mouse IgG/HRP (1:1000)
Secondary antibody incubation: 1 h at room temperature
Chemiluminescent substrate: WESTAR NOVA 2.0 (Cyanagen)
Imaging system: ChemiDoc MP Imaging system (Bio-Rad)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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