Streptococcus pyogenes M1 CRISPR-Cas9 Baculovirus-Insect Overexpression Lysate

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Streptococcus pyogenes M1 CRISPR-Cas9 Baculovirus-Insect Overexpression Lysate: Product Information

Product Description
This Streptococcus pyogenes M1 CRISPR-Cas9 overexpression lysate was created in Baculovirus-Insect Cells and intented for use as a Western blot (WB) positive control. Purification of CRISPR-Cas9 protein (Cat: 40572-A08B) from the overexpression lysate was verified.
Expression Host
Baculovirus-Insect Cells
Species
Streptococcus pyogenes M1
Sequence Information
A DNA sequence encoding the s. pyogenes serotype M1 cas9 (WP_010922251.1) (Met1-Asp1368) was expressed with a nuclear localization sequence (NLS) at the N-terminus, two NLSs and a polyhistidine tag at the C-terminus.
Molecule Mass
The recombinant s. pyogenes serotype M1 cas9 consists of 1407 amino acids and predicts a molecular mass of 163 kDa.

Streptococcus pyogenes M1 CRISPR-Cas9 Baculovirus-Insect Overexpression Lysate: Usage Guide

Preparation Method
Cell lysate was prepared by homogenization in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 min in 1 x SDS loading buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.
Lysis Buffer
Modified RIPA Lysis Buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 1% Sodium deoxycholate, 1mM PMSF.
Recommend Usage
1. Centrifuge the tube for a few seconds and ensure the pellet at the bottom of the tube. 2. Re-dissolve the pellet using 200μL pure water and boil for 2-5 min. 3. Store the lyophilized cell lysate at 4℃. After re-dissolution, recommend to aliquot it into smaller quantities and store at -80℃.
Sample Buffer
1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).
Stability & Storage
Store at 4℃ for up to twelve months from date of receipt. After re-dissolution, aliquot and store at -80℃ for up to twelve months. Avoid repeated freeze-thaw cycles.
Application
Western Blot (WB)
Optimal dilutions/concentrations should be determined by the end user.
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