Flow cytometry and quantitative Western analysis were performed as previously described.20 (link) Antibodies used in flow cytometry were the following: Tuj1 (Covance, 1:2500), MAP2 (BD Biosciences, Franklin Lakes, NJ; 1:40), glial fibrillary acidic protein (GFAP; Millipore, 1:20), Tra1–81 (Millipore, 1:500), and SSEA3 (Millipore, 1:500). Secondary antibodies were antimouse Alexa 488 (1:1,000), antirat Alexa 488 (1:1,000), or antirabbit Alexa 647 (1:1,000). The following antibodies were used in quantitative Western analysis: frataxin (MitoSciences, Eugene, OR; 1:1,000), RPL13a (Cell Signaling Technology, Danvers, MA; 1:2,000), and RNA polymerase II (Millipore; 1:2,000). Antibodies against acetylated residues of histone H3 and H4 have been described.22 (link) The following secondary antibodies were all obtained from LI-COR Biosciences (Lincoln, NE) and used at the same dilution (1:5,000): antimouse IR680, antimouse IR800, antirabbit IR680, and antirabbit IR800.
Antimouse ir680
Manufactured by LI COR
The Antimouse IR680 is a near-infrared fluorescent dye used for protein detection and quantification in western blot and other immunoassay applications. The dye has an excitation maximum at 680 nm and an emission maximum at 700 nm, making it compatible with near-infrared imaging systems.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Rna polymerase 2, by Merck Group (1 mentions)
Tra 1 81, by Merck Group (1 mentions)
Ssea3, by Merck Group (1 mentions)
Rpl13a, by Cell Signaling Technology (1 mentions)
Antimouse ir800, by LI COR (1 mentions)
Antirabbit ir800, by LI COR (1 mentions)
Image studio lite software, by LI COR (1 mentions)
Odyssey fc scanner, by LI COR (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
2 protocols using antimouse ir680
1
Comprehensive Protein Analysis Methods
2
Quantitative Dot Blot Analysis of Brain Proteins
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Protein concentration of brain tissue homogenates were assessed using the Pierce BCA Protein Assay Kit (Thermo Scientific) according to manufacturer’s instructions. Dot blotting was performed for semi-quantitative assessment of changes in protein levels, using a 96-well Bio-Rad Bio-Dot Microfiltration Unit (Bio-Rad Laboratories Ltd, UK). Samples were loaded at 2 μg of protein per well in PBS and loaded in triplicate. Samples were blotted into nitrocellulose membranes under vacuum, and the membrane was then removed and blocked in Odyssey blocking buffer (LI-COR, UK). Primary antibodies (mouse anti-gp91 1:500, BD Biosciences 611415; mouse anti-3NT, 1:750, Abcam ab61392) were diluted in Odyssey blocking buffer, incubated overnight with the membrane, washed and then incubated in secondary antibody solution (anti-mouse IR680 1:10000, LI-COR). Membranes were visualised using a LI-COR Odyssey FC scanner, and dot blots were analysed using Image Studio Lite software (LI-COR, UK). Signal intensity from each dot in the target protein channel was normalised to the loading control signal, and the results from triplicate values were averaged.
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