More information on this topic can be found in our “ Five Tips for Picking the Right Cell Disruption Method for Protein Analysis” video.Īs lipids can impact the quality of your western blots, ensure you remove fat from your samples. Ensure that you have optimized the power settings on mechanical disruptors and use pre-chilled equipment. It is important to avoid foaming during this step as it may decrease your recovery volume. To further dissociate the tissue and to shear cellular DNA, sonication of samples might be needed. Harsher disruption methods, like homogenization techniques, are required for tissues due to the higher degree of sample structure compared to cells. Five Tips for Picking the Right Cell Disruption Method for Protein Analysis. Unless otherwise indicated, these products are for research use only and are not intended for human or animal diagnostic, therapeutic or commercial use.Fig. By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. *These products may be covered by one or more Limited Use Label Licenses (see the BioLegend Catalog or our website, BioLegend products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products, reverse engineer functionally similar materials, or to provide a service to third parties without written approval of BioLegend. BioLegend will not be held responsible for patent infringement or other violations that may occur with the use of our products. Reactivity to Drosophila was only verified with the purified format. It is recommended that the reagent be titrated for optimal performance for each application. For Western blotting, the suggested use of this reagent is 0.01 - 0.1 µg/ml in human, mouse, rat and zebrafish and 0.1 - 0.5 µg/ml in Drosophila. The antibody solution should be stored undiluted between 2☌ and 8☌.Įach lot of this antibody is quality control tested by Western blotting. The antibody was purified by affinity chromatography. Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide. Enhanced chemiluminescence was used as the detection system. The blot was incubated with 0.1 µg/mL of the primary antibody overnight at 4☌, followed by incubation with HRP-labeled goat anti-rat IgG (Cat. Lane 1: Molecular weight marker Lane 2: 20 µg of Drosophila head lysate Lane 3: 20 µg of Drosophila S2 (embryonic) cell lysate. Western blot of purified anti-β-actin antibody (clone W16197A). GAPDH (poly6314, cat# 631401) antibody was used as a loading control (lower). Proteins were visualized using an HRP anti rat-IgG secondary antibody (cat# 405405) or HRP Donkey anti-rabbit IgG Antibody (cat# 406401) for GAPDH and chemiluminescence detection. Total lysates (15 µg protein) from HeLa was resolved by electrophoresis (4-20% Tris-glycine gel), transferred to nitrocellulose, and probed with 1:800 diluted (0.25 µg/mL), 1:1600 diluted (0.125 µg/mL), 1:3200 diluted (0.0625 µg/mL), 1:6400 diluted (0.03125 µg/mL) and 1:12800 diluted (0.0155 µg/mL) purified anti-β-actin antibody (upper). Total lysates (15 µg protein) from HeLa, NIH3T3, rat brain and zebra fish were resolved by electrophoresis (4-20% Tris-glycine gel), transferred to nitrocellulose, and probed with 1:2000 diluted (0.1 µg/mL) purified anti-β-actin antibody (upper).
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