Protein extracts for Western blots from cultured HEK293-P9C cells were prepared as described previously (Baker et al. 2015b (link)). Protein extracts for co-IP from HEK293 cells was prepared using HEPES-based buffer as described previously (Baker et al. 2015b (link)). For co-IP, protein lysates were diluted to 500 µL using HEPES buffer and treated with 4 µL antibody and 20 µL of Protein-A/G magnetic beads (Fisher). Immunocomplexes were incubated overnight at 4°C with rotation, washed three times with HEPES wash buffer (50 mM Hepes-KOH at pH 7.4, 150 mM NaCl, 0.4% NP-40), and eluted with 20 µL of 2× sample loading buffer (50 mM Tris-HCL at pH 6.8, 2% SDS, 10% glycerol, 100 mM DTT, 0.05% bromophenol blue) heated for 10 min at 95°C. Primary antibodies for Western blots and immunoprecipitation included anti-Flag M2 (Millipore/Sigma F3165), anti-PRDM9 (custom) (Parvanov et al. 2017 (link)), anti-HELLS (Millipore/Sigma ABD41, lot 3069868), and anti-β-Tubulin (Sigma T4026, lot 125M4884V). Secondary antibodies for Western blots included goat antimouse HRP (Bio-Rad 170-6516), goat antirabbit HRP (Bio-Rad 172-1019), and donkey antiguinea pig (Millipore AP193P).
Donkey antiguinea pig
Manufactured by Merck Group
Donkey antiguinea pig is a lab equipment product used for research purposes. It serves as a control antibody that does not bind to guinea pig antigens, providing a baseline for experiments involving guinea pig samples.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse hrp, by Bio-Rad (1 mentions)
Protein a g magnetic bead, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit hrp, by Bio-Rad (1 mentions)
Anti β tubulin, by Merck Group (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Cryostar nx50, by Thermo Fisher Scientific (1 mentions)
2 protocols using donkey antiguinea pig
1
HEK293 Cell Protein Extraction and Co-IP
2
Immunofluorescence of Apoptotic Neurons
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Frozen tissues were cut at 10 μm thickness using a cryostat (CryoStar NX50; Thermo Fisher Scientific, Rockford, IL, USA). The slices were put to a slide and rinsed three times (10 min per time) in phosphate-buffered saline (PBS, PH 7.4; # BP24384, Fisher Scientfic, Fair Lawn, NJ, USA) before being transferred to a blocking buffer for one and a half hours (PBS with 0.3% Triton x-100, 5% normal donkey serum) at room temperature. Then they were incubated in PBS 0.3% Triton x-100 solution with primary antibodies (CC3; Cell Signaling, #9661S, 1:800 and NeuN; Millipore, #AbN90P, 1:400) for 22–24 h at 4°C, followed by three-time wash (5 min per time) in PBS before being transferred to a PBS solution with secondary antibodies (Donkey anti-guinea Pig; Millipore, #AP193C, 1:500 and Donkey anti-rabbit; Invitrogen, #A21206, 1:2000) for 1-h at room temperature in the dark. Tissues were rinsed in PBS again (four times, 5 min per time) before coverslipped with DAPI mounting medium (H-1500, Vector Laboratories, Burlingame, CA, USA). Double label immunofluorescence was performed with cleaved caspase-3 (CC3) as a marker of apoptosis and NeuN as a neuron-specific marker.
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