Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing AsCpf1 (Strain BV3L6), FnCpf1 (Strain U112), Cas9 (S. pyogenes), or Cas9 (S. aureus) (+), using AsCpf1 (Strain BV3L6) Antibody (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 263
AsCpf1 (Strain BV3L6) Antibody recognizes transfected levels of total AsCpf1 (Strain BV3L6) protein. This antibody does not cross-react with Cas9 (S. pyogenes), Cas9 (S. aureus), and FnCpf1 (Strain U112) proteins.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ile57 of Acidaminococcus sp. Cpf1 (Strain BV3L6) protein. Antibodies are purified by protein A and peptide affinity chromatography.
CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) are RNA-guided nuclease effectors that are utilized for precise genome editing in mammalian systems (1). Cpf1 (CRISPR from Prevotella and Francisella) are members of the Class 2 CRISPR system (2). Class 2 CRISPR systems, such as the well characterized Cas9, rely on single-component effector proteins to mediate DNA interference (3). Cpf1 endonucleases, compared to Cas9 systems, have several unique features that increase the utility of CRISPR-based genome editing techniques: 1) Cpf1-mediated cleavage relies on a single and short CRISPR RNA (crRNA) without the requirement of a trans-activating crRNA (tracrRNA), 2) Cpf1 utilizes T-Rich protospacer adjacent motif (PAM) sequences rather than a G-Rich PAM, and 3) Cpf1 generates a staggered, rather than a blunt-ended, DNA double-stranded break (2). These features broaden the utility of using CRISPR-Cas systems for specific gene regulation and therapeutic applications. Several Cpf1 bacterial orthologs have been characterized for CRISPR-mediated mammalian genome editing (2, 4).
AsCpf1 (Strain BV3L6) is a Cpf1 enzyme that is derived from Acidaminococcus sp. BV3L6 (5, 6).
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