The cerebellum was removed and dounced in 1.5X SDS sample buffer at a concentration of 100 mg/mL. Samples were boiled for 15 mins, chilled on ice for 10 mins and insoluble material was spun out at 13,000 RPM for 3 min. 15 μL of total cell lysate/sample was run on 8–12% SDS-PAGE gels and transferred to nitrocellulose. Western blots were blocked in 5% milk, 5% NGS and 1X TBST for 1 hr. The following antibodies were used: rabbit anti-Kdm6a (1:1000, Cat #33510S, Cell Signaling Technologies), rabbit anti-H3K27me3 (1:1250, Cat #07-449, Millipore), mouse anti-Histone H3 (1:1500, Cat #05-499, Millipore) and mouse anti-actin (1:10,000, Cat #MAB1501, Millipore). Secondary antibodies were goat anti-rabbit 770 and goat anti-mouse 680 (1:1000, Biotium). Fluorescent immunoreactivity was imaged on a LI-COR Odyssey and quantified using Fiji. Kdm6a, H3K27me3 and Histone H3 were normalized to actin expression in the same lane. H3K27me3 was then normalized to Histone H3 expression in the same condition.
Mouse anti histone h3
Manufactured by Merck Group
Mouse anti-Histone H3 is a laboratory reagent used in various research applications. It is a monoclonal antibody specifically designed to detect and bind to the histone H3 protein, which is a core component of nucleosomes in eukaryotic cells. This product can be used in techniques such as Western blotting, immunoprecipitation, and immunohistochemistry to study the localization and expression of histone H3.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti h3k27me3, by Merck Group (2 mentions)
Goat anti rabbit 770, by Biotium (1 mentions)
Goat anti mouse 680, by Biotium (1 mentions)
Mouse anti actin, by Merck Group (1 mentions)
Ab8895, by Abcam (1 mentions)
B 5 1 2, by Merck Group (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Gtx124222, by GeneTex (1 mentions)
Rabbit anti h3k4me3, by Abcam (1 mentions)
Horseradish peroxidase conjugated goat anti mouse and anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Clarity western ecl kit, by Bio-Rad (1 mentions)
Goat anti mouse irdye 800 or 680, by LI COR (1 mentions)
Anti rabbit irdye 800 or 680, by LI COR (1 mentions)
Epiquik total histone extraction kit, by Epigentek (1 mentions)
Odyssey system, by LI COR (1 mentions)
2 protocols using mouse anti histone h3
1
Cerebellum Protein Expression Analysis
2
Western Blot Analysis of Proteins and Histones
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Proteins (20–40 µg) were separated on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with rabbit anti-HA tag (1:1000, C29F4, Cell Signaling, Danvers, MA), rabbit anti-KDM6B (1:1000, GTX124222, GeneTex, Irvine, CA), mouse anti-NEFM (1:200, NF-09, sc-51683, Santa Cruz Biotechnology, Dallas, TX) or mouse anti-α-tubulin (1:5000, B-5-1-2, Sigma-Aldrich). Histones were extracted using the EpiQuik total histone extraction kit (EpiGentek, Farmingdale, NY) and analyzed by immunoblotting using mouse anti-histone H3 (1:1000, 05-499, Millipore, Burlington, MA), rabbit anti-H3K27me3 (1:1000, 07-449, Millipore) or rabbit anti-H3K4me3 (1:1000, ab8895, Abcam, Cambridge, MA). Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit IgG (Santa Cruz Biotechnology) were used as secondary antibodies. Proteins were visualized using a Clarity Western ECL kit (#1705061, Bio-RAD, Hercules, CA). For visualization using the Odyssey system (LI-COR, Lincoln, NE), goat anti-mouse IRDye 800 or 680 and anti-rabbit IRDye 800 or 680 from LI-COR were used as secondary antibodies.
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