Editor's Note: All antibody reviews will appear in protocol format.
Primary Antibody: Anti-phospho-Ser/Thr-Pro, MPM-2 (Mitotic protein monoclonal #2)
Antigen/Epitope: Phosphorylated serines and threonines when followed by a proline
Supplier and catalog number: EMD Millipore 05-368MG (previously Upstate Biotechnology)
Sample: 30-50 mouse oocytes
Secondary Antibody: Affinity-Purified Fluoresceinated Donkey Anti-Mouse IgG
Supplier and catalog number: Jackson ImmunoResearch 715-095-150
Block: 2% BSA, 2% Powdered Milk, 2% Normal Goat Serum in 0.1 M Glycine and 0.01% Triton X-100
Incubation time: Overnight
Incubation temperature: 4°C
Primary Antibody Incubation
Dilution used: 1/100
Diluent composition: 2% BSA, 2% Powdered Milk, 2% Normal Goat Serum in 0.1 M Glycine and 0.01% Triton X-100
Incubation time: 90 min
Incubation temperature: 37°C
Secondary Antibody Incubation
Dilution used: 1/100
Diluent composition: 2% BSA, 2% Powdered Milk, 2% Normal Goat Serum in 0.1 M Glycine and 0.01% Triton X-100
Incubation time: 1 hr
Incubation temperature: 37°C
Detection Method: Zeiss IM-35 inverted microscope using 50-W mercury arc lamp and a 40x Neofluor objective. Digital images were collected with a Hamamatsu Orca ER digital camera (model #C4742-95) interfaced with a MetaMorph Imaging System (Molecular Devices, Sunnyvale, CA).
Results Summary: The image below shows an M-I mouse oocyte. (Blue shows chromatin staining used during the mounting procedure.)
Additional notes/tips: I've also run these experiments several times in mouse cumulus cells and in these samples, the dilution of primary antibody can range from 1:100 to 1:500 although the best results are obtained with the lesser dilution.
(Note: The image at the top of this page is from EMD Millipore.)