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重组嗜酸细菌Cas12b蛋白
AapCas12属于V型CRISPR效应子CRISPR-Cas12b/C2c1,具有很高的热稳定性,可广泛应用。在大肠杆菌中表达的与Alicyclobacillus acidiphilus Cas12b(V型CRISPR相关蛋白Cas12b)相对应的全长重组蛋白。
Molecular Weight
~128kD
Assay Conditions
Varying amounts of AapCas12b activity was measured using a CRISPR-based fluorescent reporter assay for optimal results. Target DNA cutting and indiscriminate single stranded DNA collateral cleavage was activated using RNA-guided DNA Binding to Cas12. Emission of fluorescent signal is due to the degradation of ssDNA reporters upon cleavage.
Active Cas12 was thawed on ice while 1X Endonuclease Buffer containing 10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10 mM MgCl2, and 0.1 mg/ml BSA, guide RNA (custom designed crRNA), ds DNA activator (complementary sequence to crRNA and a PAM sequence specific for Cas enzyme) and FQ-ssDNA substrate (labeled with fluorophore and a quencher) were equilibrated to room temperature. Next three working solutions of Active Cas12 (4X final concentration) guide RNA (4X final concentration) and activator/reporter mix containing ds DNA activator and ssDNA reporter (2X final concentration), were prepared using 1X Endonuclease Buffer. 10 μl of 4X active Cas12 and 10 μl of 4x guide RNA were then preincubated in half the area of a solid black 96-well plate for 10 minutes at room temperature. After preincubation, 20 μl of 2X activator/reporter mix was added to plate and placed on shaking incubator for 1 min. The plate was then sealed and incubated at 37°C for 10-30 minutes. Plate was then equilibrated to room temperature, plate sealer removed and fluorescence read on a microplate reader. Negative control was measured by replacing enzyme working solution with equal volume of assay buffer.
Applications
Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling.
Storage and Stability
Aliquot to avoid repeated freezing and thawing and store at -70°C. Aliquots are stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
Source
Recombinant, E. coli
Purity
≥75%
Concentration
As Reported
Form
Supplied as a liquid in 50mM sodium phosphate, pH 7.5, 300mM sodium chloride, 1mM DTT, 10% glycerol
Grade
Purified
Accession Number
WP_067623834
Molecular Weight
128
产品组分
内容 |
型号 |
规格 |
储存温度 |
重组嗜酸细菌Cas12b蛋白 | 045812 | 20ug | -70°C |
操作手册 |
1 | 1 |
常温 |
注意事项
保存建议 | 厂家推荐蓝冰运输。当您收到产品后,按照说明书建议保存于-20°C。 |
FAQ
1. Ocampo Lyra J.S.P., Madi K., Maeda C.T., Savino W. (1993) Thymic extracellular matrix in human malnutrition. J. Pathol. 1993, 171, 231-236.
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