Tissues were washed exhaustively with PBS to remove blood and other debris. A lysate was prepared by homogenizing the tissue and washing the cells in cold PBS. Washed cells were incubated at 4° C in modified RIPA buffer containing 150 mM sodium chloride, 50 mM Tris Cl, pH 7.4, 1 mM EDTA, 1.0% NP-40, 0.5% sodium deoxycholic acid and 0.1% SDS 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 µM Aprotinin, 5 µM Bestatin, 1.5 µM E-64, 2 µM Leupeptin Hemisulfate and 1 µM Pepstatin A). The following phosphatase inhibitors were also added: 1 mM NaF and 1 mM Na3VO4. Cell debris was removed by centrifugation and 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.
Conjugation
Unconjugated
Hazard Information
Non-Toxic
Source
Chicken
Biological Origin
Chicken
Storage
Store vial at -70° C or COLDER. For extended storage, aliquot contents to minimize freeze/thaw cycles.
Buffer/Preservatives
Preservative: None. Stabilizer: 10% (v/v) Glycerol. 1X SDS-PAGE Sample Buffer (62.5 mM Tris HCl, 2% SDS, 10% Glycerol and 0.005% bromophenol blue, pH 6.8)
Alternative names
Normal Chicken Spinal Cord Whole Cell Lysate, chic
Note
For research use only
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 95°C for 5 minutes and rapidly cool. If dissociating conditions are desired, add reducing agent prior to heating. The recommended loading volume per lane is 5-15 μl depending on the size format of your gel.
