MEF Whole Cell Lysate

Western Blot: MEF Whole Cell Lysate [NB800-PC10] - Coomassie stain of 0.02 ml of NB 800-PC10 separated on a 4-20% gradient gel under non-reducing conditions.

• Reactivity: Mu
• Applications: WB, PAGE

MEF Whole Cell Lysate Summary

• Description: Store vial at -70C or COLDER. For extended storage, aliquot contents to minimize freeze/thaw cycles.
• Preparation Method: The cells were grown in RPMI supplemented with 10% FBS (Fetal Bovine Serum). The lysate was prepared by first washing the cells in PBS. Washed cells are then incubated on ice in modified RIPA buffer containing 150 mM sodium chloride, 50 mM Tris Cl, pH 7.4, 1 mM EDTA, 1.0% NP-40, 0.25% sodium deoxycholic acid to lyse the cells. Protein integrity is 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 and 1 uM Pepstatin A). Phosphatase inhibitors include 1 mM Sodium Fluoride and 1mM Na3VO4. Cell debris was removed by membrane filtration. Protein concentration was determined by Lowry assay using a commercially available kit. The 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: 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

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.