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Goat anti mouse 680

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Goat anti-mouse 680 is a secondary antibody conjugated with a fluorescent dye. It is designed to detect and bind to mouse primary antibodies, allowing for their visualization in various immunoassays and imaging applications.

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Lab products found in correlation

Goat anti rabbit 770, by Biotium (3 mentions) Anti ldlr, by Abcam (3 mentions) Anti actin, by Merck Group (3 mentions) Anti gapdh, by Cell Signaling Technology (3 mentions) Clarity western ecl substrate, by Bio-Rad (3 mentions) Chemidoc xr, by Bio-Rad (3 mentions) Mouse anti actin, by Merck Group (3 mentions) Mab1501, by Merck Group (3 mentions) F3165, by Merck Group (2 mentions) Mouse anti flag, by Merck Group (2 mentions) Rabbit anti h3k27me3, by Merck Group (1 mentions) Mouse anti histone h3, by Merck Group (1 mentions) Ab50005, by Abcam (1 mentions) Sc 25778, by Santa Cruz Biotechnology (1 mentions) Odyssey system, by LI COR (1 mentions)

8 protocols using goat anti mouse 680

1

Cerebellum Protein Expression Analysis

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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.
Wijayatunge R., Liu F., Shpargel K.B., Wayne N.J., Chan U., Boua J.V., Magnuson T, & West A.E. (2017). The histone demethylase Kdm6b regulates a mature gene expression program in differentiating cerebellar granule neurons. Molecular and cellular neurosciences, 87, 4-17.
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2

Quantitative Western Blot Analysis

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Protein from tissue was isolated using a Biomasher II mortar and pestle tubes in conjunction with RIPA or TGH buffer. Protein was quantified using BCA assay. Novex NuPage 4-12% Bis-Tris gels were run for 45 minutes in MES running buffer and transferred onto nitrocellulose membranes for an hour at 4 °C in towbin’s buffer with 20% MeOH. Membranes were blocked for non-specific binding overnight in 5% milk in TBS-T. 1° Antibodies were incubated in 5% milk in TBS-T and used at the following manner: Anti-Cas9 (EnCor Biotechnology, Cat#MCA-3F, 1:2000) at room temp for 2-3 hours, Anti-Gapdh (Cell Signaling, Cat#2118L, 1:5000), Anti-LDLR (Abcam, Cat#ab52818, 1:1000), and anti-Actin (EMD Millipore, Cat#MAB1501, 1:5000) overnight at 4°C. After washing, secondary antibodies (Anti-rabbit HRP (Sigma, Cat#A6154, 1:5000) and Anti-Mouse HRP (Santa Cruz, Cat#sc-2005, 1:5000) were incubated in 3% BSA in TBS-T. Membranes were visualized using Clarity western ECL substrate (Cat#1705061) from BioRad and imaged using a BioRad ChemiDoc XRS+. For neurons, cells were lysed directly in 1X RIPA buffer and blots were incubated with goat anti-mouse 680 (Biotium, Cat#20253, 1:5000). Fluorescent immunoreactivity was imaged on a LICOR Odyssey and analyzed with LiCor Image Studio v4.0.
Gemberling M.P., Siklenka K., Rodriguez E., Tonn-Eisinger K.R., Barrera A., Liu F., Kantor A., Li L., Cigliola V., Hazlett M.F., Williams C.A., Bartelt L.C., Madigan V.J., Bodle J.C., Daniels H., Rouse D.C., Hilton I.B., Asokan A., Ciofani M., Poss K.D., Reddy T.E., West A.E, & Gersbach C.A. (2021). Transgenic mice for in vivo epigenome editing with CRISPR-based systems. Nature methods, 18(8), 965-974.
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3

MeCP2 Expression in Rat Brain Regions

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To harvest tissue for western blotting in Experiment 1, rats were deeply anesthetized with isofluorane, followed by rapid decapitation. The brains were rapidly dissected and placed in OCT for snap-freezing in an isopentane/dry ice bath, then stored at -80°. Coronal sections from frozen brains were cut on a freezing microtome, and mPFC and striatum were dissected by hand, weighed, and lysed in SDS sample buffer to a final concentration of 100 mg/mL. One mg total cell lysate/sample was run on SDS-PAGE and transferred to nitrocellulose for western blotting with the following antibodies: mouse anti-MeCP2 1:1,000 (Ab50005, Abcam), rabbit anti-GAPDH 1:2,000 (sc-25778, Santa Cruz), goat anti-rabbit 770 and goat anti-mouse 680 1:1,000 (Biotium). Fluorescent immunoreactivity was imaged on a LI-COR Odyssey system and quantified using ImageJ. MeCP2 expression was normalized to GAPDH expression in the same lane.
Deng J.V., Orsini C.A., Shimp K.G, & Setlow B. (2017). MeCP2 expression in a rat model of risky decision making. Neuroscience, 369, 212-221.
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4

Western Blot Analysis of MeCP2 Phosphorylation

Cited 1 time
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Brain regions were dounced in 20µL 1% SDS and boiled for 3 min. They were then adjusted to 100µL in RIPA buffer (10mM Tris pH7.5, 1mM EDTA, 1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 150mM NaCl), and insoluble material was spun out at 13,000 RPM for 3 min. Samples were subjected to BCA protein assay, and 30µg of total cell lysate/sample was run on 8% SDS-PAGE and transferred to nitrocellulose for Western blotting with the following primary antibodies: mouse anti-actin (1:10,000; MAB1501; Millipore), rabbit anti-phospho-Ser421 MeCP2 (1:1,000; Deng et al., 2010 (link)), mouse anti-MeCP2 (Ab5005 Abcam). 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 ImageJ. MeCP2 was normalized to actin expression in the same lane. Normalized MeCP2 levels in WT mice for each single brain region were scaled to a value of 100, and MeCP2 KI levels are graphed relative to WT in that brain region.
Zannas A.S., Kim J.H, & West A.E. (2017). Regulation and function of MeCP2 Ser421 phosphorylation in U50488-induced conditioned place aversion in mice. Psychopharmacology, 234(6), 913-923.
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5

Western Blot Analysis of Neuro2a Cells

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Neuro2a cells were lysed directly into 2x SDS sample lysis buffer. Samples were sonicated, boiled for 5 min, chilled on ice, then centrifuged at 13,000 RPM for 3 minutes to remove insoluble material. 10uL of total cell lysate was run on a 8%–12% SDS-PAGE gel and transferred to nitrocellulose. Western blots were blocked in 5% non-fat dry milk in TBST. Primary antibodies included mouse anti-actin (1:20000, MAB1501, EMD Millipore) and mouse anti-FLAG (1:4000, F3165, Sigma). After thorough washing with TBST, blots were incubated with goat anti-mouse 680 (1:5000, cat #20253, Biotium). Fluorescent immunoreactivity was imaged on a LICOR Odyssey.
Chen L.F., Lin Y.T., Gallegos D.A., Hazlett M.F., Gómez-Schiavon M., Yang M.G., Kalmeta B., Zhou A.S., Holtzman L., Gersbach C.A., Grandl J., Buchler N.E, & West A.E. (2019). Enhancer Histone Acetylation Modulates Transcriptional Bursting Dynamics of Neuronal Activity-Inducible Genes. Cell reports, 26(5), 1174-1188.e5.
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6

Western Blot Analysis of Neuro2a Cells

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Neuro2a cells were lysed directly into 2x SDS sample lysis buffer. Samples were sonicated, boiled for 5 min, chilled on ice, then centrifuged at 13,000 RPM for 3 minutes to remove insoluble material. 10uL of total cell lysate was run on a 8%–12% SDS-PAGE gel and transferred to nitrocellulose. Western blots were blocked in 5% non-fat dry milk in TBST. Primary antibodies included mouse anti-actin (1:20000, MAB1501, EMD Millipore) and mouse anti-FLAG (1:4000, F3165, Sigma). After thorough washing with TBST, blots were incubated with goat anti-mouse 680 (1:5000, cat #20253, Biotium). Fluorescent immunoreactivity was imaged on a LICOR Odyssey.
Chen L.F., Lin Y.T., Gallegos D.A., Hazlett M.F., Gómez-Schiavon M., Yang M.G., Kalmeta B., Zhou A.S., Holtzman L., Gersbach C.A., Grandl J., Buchler N.E, & West A.E. (2019). Enhancer Histone Acetylation Modulates Transcriptional Bursting Dynamics of Neuronal Activity-Inducible Genes. Cell reports, 26(5), 1174-1188.e5.
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7

Quantitative Western Blot Analysis

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Protein from tissue was isolated using a Biomasher II mortar and pestle tubes in conjunction with RIPA or TGH buffer. Protein was quantified using BCA assay. Novex NuPage 4-12% Bis-Tris gels were run for 45 minutes in MES running buffer and transferred onto nitrocellulose membranes for an hour at 4 °C in towbin’s buffer with 20% MeOH. Membranes were blocked for non-specific binding overnight in 5% milk in TBS-T. 1° Antibodies were incubated in 5% milk in TBS-T and used at the following manner: Anti-Cas9 (EnCor Biotechnology, Cat#MCA-3F, 1:2000) at room temp for 2-3 hours, Anti-Gapdh (Cell Signaling, Cat#2118L, 1:5000), Anti-LDLR (Abcam, Cat#ab52818, 1:1000), and anti-Actin (EMD Millipore, Cat#MAB1501, 1:5000) overnight at 4°C. After washing, secondary antibodies (Anti-rabbit HRP (Sigma, Cat#A6154, 1:5000) and Anti-Mouse HRP (Santa Cruz, Cat#sc-2005, 1:5000) were incubated in 3% BSA in TBS-T. Membranes were visualized using Clarity western ECL substrate (Cat#1705061) from BioRad and imaged using a BioRad ChemiDoc XRS+. For neurons, cells were lysed directly in 1X RIPA buffer and blots were incubated with goat anti-mouse 680 (Biotium, Cat#20253, 1:5000). Fluorescent immunoreactivity was imaged on a LICOR Odyssey and analyzed with LiCor Image Studio v4.0.
Gemberling M.P., Siklenka K., Rodriguez E., Tonn-Eisinger K.R., Barrera A., Liu F., Kantor A., Li L., Cigliola V., Hazlett M.F., Williams C.A., Bartelt L.C., Madigan V.J., Bodle J.C., Daniels H., Rouse D.C., Hilton I.B., Asokan A., Ciofani M., Poss K.D., Reddy T.E., West A.E, & Gersbach C.A. (2021). Transgenic mice for in vivo epigenome editing with CRISPR-based systems. Nature methods, 18(8), 965-974.
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8

Western Blot Analysis of Protein Samples

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Protein from tissue was isolated using a Biomasher II mortar and pestle tubes in conjunction with RIPA or TGH buffer. Protein was quantified using BCA assay. Novex NuPage 4-12% Bis-Tris gels were run for 45 minutes in MES running buffer and transferred onto nitrocellulose membranes for an hour at 4 ˚C in towbin's buffer with 20% MeOH. Membranes were blocked for non-specific binding overnight in 5% milk in TBS-T. 1˚ Antibodies were incubated in 5% milk in TBS-T and used at the following manner: Anti-Cas9 (EnCor Biotechnology/Cat#MCA-3F)-1:2000 at room temp for 2-3 hours, Anti-Gapdh (Cell Signaling/Cat#2118L) 1:5000, Anti-LDLR (Abcam/Cat# ab52818)-1:1000, and anti-Actin (EMD Millipore/Cat#MAB1501) overnight at 4˚C. After washing, secondary antibodies (Anti-rabbit HRP (Sigma/Cat#A6154) and Anti-Mouse HRP (Santa Cruz/Cat#sc-2005) were incubated in 3% BSA in TBS-T at 1:5000. Membranes were visualized using Clarity western ECL substrate (Cat#1705061) from BioRad and imaged using a BioRad ChemiDoc XRS+. For neurons, cells were lysed directly in 1X RIPA buffer and blots were incubated with goat anti-mouse 680 (1:5000, cat #20253, Biotium). Fluorescent immunoreactivity was imaged on a LICOR Odyssey.
Gemberling M., Siklenka K., Rodriguez E., Eisinger K.R.T., Barrera A., Liu F., Kantor A., Li L., Cigliola V., Hazlett M.F., Williams C., Bartelt L.C., Madigan V.J., Bodle J.C., Daniels H., Rouse D., Hilton I.B., Asokan A., Ciofani M., Poss K.D., Reddy T.E., West A.E., & Gersbach C.A. (2021). Transgenic mice for in vivo epigenome editing with CRISPR-based systems.
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