CHO K1 Whole Cell Lysate

Western Blot: CHO K1 Whole Cell Lysate [NB800-PC13] - Detection of alpha tubulin in lane 1. CHO/K1 Whole Cell Lysate (10 g) was run on a 4-20% gel, then transferred to 0.45 um nitrocellulose. After blocking with 1% BSA-TTBS 30 min at 20C, primary antibody was used at 1:1000 overnight at 4C. Anti-Rabbit IgG (H&L) (GOAT) antibody IRDye800CW secondary antibody was used at 1:20,000 in Blocking Buffer for Fluorescent Western Blotting and imaged on the LiCor Odyssey imaging system. Arrow indicates correct 50 kDa molecular weight position expected for alpha tubulin.

Western Blot: CHO K1 Whole Cell Lysate [NB800-PC13] - Coomassie stained SDS-PAGE of 35 ul of CHO Whole Cell Lysate (Ready-to-Use) separated in a 4-20% gradient gel under reducing conditions (lane 2). Molecular weight standards are shown in lane 1.

• Reactivity: ChHa
• Applications: WB, PAGE

CHO K1 Whole Cell Lysate Summary

• Preparation Method: The cells were grown in Ham's F12 medium containing 2 mM L-Gln and 1.5 g/L sodium bicarbonate supplemented with 10% FBS (Fetal Bovine Serum). Cells were washed with PBS and then incubated on ice in modified RIPA buffer, containing 150 mM sodium chloride, 50 mM Tris HCl, pH 7.4, 1 mM EDTA, 1.0% NP-40, 0.5% sodium deoxycholic acid , 0.1% SDS and 0.01% (w/v) sodium azide to lyse the cells. Protein integrity was ensured using a cocktail of protease inhibitors with broad specificity for the inhibition of aspartic, cysteine, and serine proteases as well as aminopeptidases (0.1 mM AEBSF HCl, 0.08 uM Aprotinin, 5 uM Bestatin, 1.5 uM E-64, 2 uM Leupeptin Hemisulfate, 1 uM Pepstatin A). Phosphatase inhibitors 1 mM NaF and 1 mM Na3VO4 were also added. Cell debris was removed by centrifugation. Protein concentration was determined by a modified Lowry assay using a commercially available kit. Protein concentration was adjusted to 2 mg/ml and then an equal volume of 2X SDS-PAGE sample buffer was added.
• Purity: Multi-step

Applications/Dilutions

• Dilutions:
  • SDS-Page
  • Western Blot
• Application Notes: This product has been tested by SDS-PAGE and western blot. Ready-to-use lysates are especially prepared as positive controls for separation by SDS-PAGE and subsequent western blot analysis. Lysates are prepared in denaturing buffer WITHOUT dissociating agents (i.e. no 2-mercaptoethanol or dithiothreitol has been added). Heat lysate to 95C for 5 minutes and rapidly cool. If dissociating conditions are desired, add reducing agent prior to heating. The recommended loading volume per lane is 10-20 ul depending on the size format of your gel.

Packaging, Storage & Formulations

• Storage: Store at -70C. Avoid freeze-thaw cycles.
• Buffer: 1X SDS-PAGE Sample Buffer (62.5 mM Tris HCl, 2% SDS, 10% Glycerol and 0.005% bromophenol blue, pH 6.8) 10% (v/v) Glycerol
• Preservative: No Preservative
• Purity: Multi-step

Lysate Details for Array

• Type: Cell
• Subcellular Fraction: Whole

Notes

Store vial at -70C or COLDER. For extended storage, aliquot contents to minimize freeze/thaw cycles.

Background

This ready-to-use whole cell lysate is derived from a cell line or tissue using highly refined extraction protocols to ensure exceptionally high quality, protein integrity and lot-to-lot reproducibility. All extracts are tested by SDS-PAGE using 4-20% gradient gels and immunoblot analysis using antibodies to key cell signaling components to confirm the presence of both high molecular weight and low molecular weight proteins.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.