Antibodies and Chemicals for Lysosome Analysis

The following commercially available antibodies were used: anti-Rab5 (ab31261, abcam), anti-GABARAP for Drosophila Atg8a (PM037, MBL), anti-Drosophila LAMP1 (ab30687, abcam), anti-human LAMP1 (AB2296838, DSHB), anti-cathepsin D (sc-6487, Santa Cruz), anti-Brp (AB2314866, DSHB), anti-cathepsin L (MAB 22591, R&D systems)^{44 (link),63}, anti-Ref(2)P²⁸, anti-elav (AB528217, DSHB), anti-ATP5α (ab14748, abcam), Cy5-conjugated anti-HRP (123175021, Jackson ImmunoLab), and secondary antibodies conjugated to Alexa 488/568/647 (Thermo Fisher Scientific), or near infrared (IR) dye 700/800 (Rockland). The following chemicals were used in the study: N-acetylcysteine amide (A0737, Sigma-Aldrich), N-(2-mercaptopropionyl)glycine (M6635, Sigma-Aldrich), putrescine (51799, Sigma-Aldrich), spermidine (S0266, Sigma-Aldrich), spermine (s4264, Sigma-Aldrich), bis(hexamethylene)triamine (421960, Sigma-Aldrich), DHE (D11347, Thermo Fisher Scientific), LysoTracker^® Red DND-99 (L7528, Thermo Fisher Scientific), LysoSensor™ Yellow/Blue DND-160 (L7545, Thermo Fisher Scientific).

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Li C., Brazill J.M., Liu S., Bello C., Zhu Y., Morimoto M., Cascio L., Pauly R., Diaz-Perez Z., Malicdan M.C., Wang H., Boccuto L., Schwartz C.E., Gahl W.A., Boerkoel C.F, & Zhai R.G. (2017). Spermine synthase deficiency causes lysosomal dysfunction and oxidative stress in models of Snyder-Robinson syndrome. Nature Communications, 8, 1257.

Publication 2017

ab31261 ab30687 MAB 22591 ab14748 IR) dye 700/800 N-acetylcysteine amide N-(2-mercaptopropionyl)glycine putrescine spermidine spermine bis(hexamethylene)triamine LysoTracker® Red DND-99 LysoSensor™ Yellow/Blue DND-160

Alexa 568 Anti d Antibodies Cathepsin Cathepsin d Cathepsin l Drosophila Hexamethylene Human Lamp1 Lysosensor yellow blue Lysotracker Mercaptopropionyl glycine N acetylcysteine amide Putrescine Red dnd 99 Spermidine Spermine

Corresponding Organization :

Other organizations : University of Miami, Yantai University, National Human Genome Research Institute, Greenwood Genetic Center

Top 5 similar protocols

Variable analysis

independent variables

N-acetylcysteine amide
N-(2-mercaptopropionyl)glycine
Putrescine
Spermidine
Spermine
Bis(hexamethylene)triamine

dependent variables

GABARAP
Drosophila LAMP1
Human LAMP1
Cathepsin D
Cathepsin L
Ref(2)P
ATP5α

control variables

Cy5-conjugated anti-HRP
Secondary antibodies conjugated to Alexa 488/568/647 or near infrared (IR) dye 700/800
LysoTracker® Red DND-99
LysoSensor™ Yellow/Blue DND-160

positive control

Anti-Ref(2)P

negative control

Not specified

Annotations

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