Quantifying Cell-Specific Protein Expression

Samples obtained from all protocols were analyzed by Western blots with 4-12% SDS-PAGE (Invitrogen, NP0322BOX). Western blot assays were performed as described previously (Sherwood et al., 2010 (link)). The following primary antibodies were used for immunoblotting: anti-LDH-A (1/1000, Santa Cruz, sc- 27230), anti-LDH-B (1/500, Novus Biologicals, NB100-79987), and anti-LDH (1/1000, Santa Cruz, sc-133123). Antibodies were characterized in detail before being used for research (see the Supplemental Material). The blots were also probed with antibodies against cell-specific marker proteins: anti-GFAP (glial marker, 1/1000, Santa Cruz, sc-9065) and anti-SYP (presynaptic neuronal marker, 1/1000, Abcam, ab14692). For detection of antibodies, appropriate peroxidase-conjugated secondary antibodies were used in conjunction with enhanced chemiluminescence (Amersham Pharmacia Biosciences) to obtain images saved on film. The signals were quantitatively evaluated with Scion Image software. Equal protein loading was confirmed with anti-β-actin antibody (1/1000, Santa Cruz Biotechnology, sc-1616). Protein bands were scanned using an EPSON Perfection 4870 Photo scanner and quantitatively analyzed by Scion Image software.

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Duka T., Anderson S.M., Collins Z., Raghanti M.A., Ely J.J., Hof P.R., Wildman D.E., Goodman M., Grossman L.I, & Sherwood C.C. (2014). SYNAPTOSOMAL LACTATE DEHYDROGENASE ISOENZYME COMPOSITION IS SHIFTED TOWARD AEROBIC FORMS IN PRIMATE BRAIN EVOLUTION. Brain, behavior and evolution, 83, 216-230.

Publication 2014

NP0322BOX anti-LDH-A anti-LDH anti-GFAP anti-SYP Scion Image software anti-β-actin antibody

Actin Anti antibody Antibodies Biologicals Cell Chemiluminescence Gfap Glial Ldh 1 Ldh a Neuronal Novus Peroxidase Protein Sds page Western blot assays

Corresponding Organization :

Other organizations : George Washington University, Kent State University, United States Air Force Academy, Icahn School of Medicine at Mount Sinai, Laboratoire d’immunologie intégrative du cancer, Wayne State University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein expression levels of LDH-A, LDH-B, and total LDH measured by Western blot

control variables

Protein loading was confirmed using anti-β-actin antibody
Cell-specific marker proteins GFAP (glial marker) and SYP (presynaptic neuronal marker) were used

controls

Positive control: None specified
Negative control: None specified

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