Purification and Labeling of Cas9 Variants
Corresponding Organization : Lawrence Berkeley National Laboratory
Variable analysis
- WT Cas9
- DCas9 (D10A/H840A)
- Cas9_HNH-1 (C80S/S355C/C574S/S867C)
- DCas9_HNH-1 (D10A/C80S/S355C/C574S/H840A/S867C)
- Cas9_HNH-2 (C80S/C574S/S867C/N1054C)
- Steady-state FRET measurements
- Dynamic FRET measurements
- Cas9 gel filtration buffer [20 mM tris-HCl (pH 7.5), 200 mM KCl, 5% glycerol, and 1 mM tris(2-carboxyethyl)phosphine (TCEP)]
- Labeling reactions contained 10 μM Cas9, 200 μM Cy3-maleimide, and 400 μM Cy5-maleimide (GE Healthcare)
- For steady-state measurements, regular cyanine dyes (GE Healthcare) were used
- For dynamic FRET measurements, Cas9 was labeled with Cy3 and Cy5 derivatives, LD550-MAL and LD650-MAL (Lumidyne Technologies)
Annotations
Based on most similar protocols
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!