nativepagetm cathode buffer additive  (Bio-Rad)

Journal: PNAS Nexus

Article Title: APOE3 astrocytes can rescue lipid abnormalities and dystrophic neurites of APOE4 human neurons

doi: 10.1093/pnasnexus/pgag053

Figure Lengend Snippet: APOE3 astrocytes have higher levels of APOE and higher levels of large APOE particles at baseline and upon NPC1 inhibition. Three isogenic APOE3 and APOE4 hiPSC-derived neuron−astrocyte (iN + hAs) cocultures were maintained for 1 week and treated with either the Veh (PBS) or the NPC1-inhibitor U18666A (NPC1 (−); 10 µg/mL) for 3 days. A) Representative native PAGE followed by APOE western blot showing large and small APOE particles in the culture media. B) Quantification of large and small APOE particle band intensity from native PAGE blots. C) Total APOE levels measured in culture media using an APOE ELISA assay. D) Representative western blot showing total APOE levels in iN + hAs lysates. E) Quantification of APOE band intensity from western blots. APOE levels were normalized to total protein levels. Data for each condition were averaged from at least three independent experiments (full differentiation of all isogenic lines) per isogenic line in B and C, and from two independent experiments per isogenic line in E. Each data point represents an isogenic line in B, C, and E. Statistical analysis was performed using two-way ANOVA with pairwise matching. Significant differences ( P < 0.05) are indicated on the graphs.

Article Snippet: Media samples were mixed with Native Sample Buffer (Bio-Rad, #1610738) supplemented with G-250 Sample Additive (Invitrogen, #BN2004) and run on 4–20% polyacrylamide tris-glycine gels in the absence of sodium dodecyl sulfate, reducing agents or sample boiling, in tris-glycine buffer supplemented with 1:20 native PAGE Cathode Buffer Additive (Invitrogen, #BN2002), at 150 V for 15 min.

Techniques: Inhibition, Derivative Assay, Clear Native PAGE, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Pharmaceuticals

Article Title: Bee Venom Proteins Enhance Proton Absorption by Membranes Composed of Phospholipids of the Myelin Sheath and Endoplasmic Reticulum: Pharmacological Relevance

doi: 10.3390/ph18091334

Figure Lengend Snippet: Isoelectic focusing (IEF) of the bee venom fractions 1–7 in Ready Gel Precast Gels with ampholytes of pH gradient from 3.0 to 10.5. A multi-sample horizontal IEF gel was employed with 11 lanes containing the seven bee venom fractions and four unrelated samples. For presentation in , the lanes of the seven bee venom fractions were excised from the original IEF gel photograph and are arranged adjacently to facilitate comparative analysis. Lane 1 includes tube 3–6, lane 2 includes tubes 7–9, lane 3 includes tubes 10–11, lane 4 includes tubes 12–13, lane 5 includes tubes 14–16, lane 6 includes tubes 27–30, and lane 7 includes tubes 31–36, as shown in . The standard p I markers (FMC Corporation, Rockland, ME, USA) sample of a p I range from 4.65 to 10.6 is applied on the unmarked lane on the far left.

Article Snippet: The following materials and chemicals were used in this study: Phospholipids—phosphatidylserine, sphingomyelin, phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine—were purified from the rat liver (see the Preparations section below), Dichloro-diphenyl-trichloroethane (DDT), Sephadex G-25 and CM Sephadex C-50 (Nanjing Duly Biotech Co., Ltd., Nanjing, China), Tris(hydroxymethyl)aminomethane (Tris) 10 M pH 8.5 buffer, 1.0 M Tris-HCl pH 6.8 with 0.4% SDS buffer, Bromo-phenol Blue (Thomas Scientific, Swedesboro, NJ, USA); Mini-PROTEAN TGX precast gels (8% density), Isoelectric Focusing Gel Sample Buffer (IEF Gel), Ready Gel Precast Gels with ampholytes making pH gradient 3–10.5, 10× IEF Anode Buffer, 10× IEF Cathode Buffer (Bio-Rad Laboratories Co., Ltd., Shanghai, China), IEF p I 4.65–10.6 range protein markers for IEF (Shanghai Yeyuan Biotechnology Co., Ltd., Shanghai, China), Sodium Dodecyl Sulfate (SDS), 50× TAE (Tris-acetate-EDTA, pH 8.3) buffer, Coomassie Brilliant Blue-R-250, low-molecular-weight markers for SDS-PAGE (Thermo Fisher Scientific Inc., Shanghai, China), lyophilized bee venom (Sigma Aldrich, Saint Louis, MO, USA), 3.5 kDa cutoff dialysis tubing (Sigma Aldrich, Saint Louis, MO, USA), research grade Glycine (Asiamerica Group, Inc., Westwood, NJ, USA); Deionized-Distilled water (dd-H 2 O) (XiZhiMeng Co., Ltd., Shanghai, China).

Techniques: