Knock-out Cell Line Validated Antibodies
Specificity is an essential property of antibodies and antibody specificity is top on the minds of today's researchers. There are a myriad of technologies for validating antibody specificity and various technologies are often used in combination with one another to help assess antibody specificity. Gene knock-out (KO) technology such as KO cell lines is emerging is as one of the most trusted methods for validating antibody specificity. Antibodies validated by gene KO technology are highly valued by researchers. MyBioSource, Inc. is pleased to offer a growing portfolio of antibodies validated with gene KO technology.
Generating KO Cell Lines
Among our portfolio are antibodies validated with KO cell lines generated by combining CRISPR/Cas9 technology and lentivirus/adenovirus transfection platforms. The cartoon illustrates how the CRISPR/Cas9 technology was combined with the lentivirus infection system to transfect target cells.
Antibody Validation with KO Cell Lines
KO cell line generation
- 1. Guide sequences of of 20nt and their complementary sequences were obtained by chemical synthesis. LentiCRISPRv2 recombinant vectors containing sgRNA sequence were constructed
- 2. The lentiCRISPRv2 recombinant vector and the lentiviral packaging plasmid were co-transfected into 293T cells. The virus particles were collected and infected into MCF-7 cells. Stably transfected cells were obtained using puromycin.
- 3. Cas9 and sgRNA (single guide RNA) can be expressed stably and the Cas9-gRNA complex was synthesized in cells. The complex was translocated into the nucleus to complete the shearing of sgRNA's complementary DNA fragments. The diagram shows gene sequencing of site-specific mutagenesis.
Western blot (WB) analysis
The BECN1 antibody (MBS2028535) was tested by WB with wild-type untreated MCF-7 (NULL-1, -2) BECN1 KO (BECN1-1, -2, -3) MCF-7 cell lysates. A GAPDH antibody (MBS2004775) was used as a housekeeping control.
The WB results show that BECN1 is expressed in untreated, wild-type MCF-7 cells. They also show that the expression of BECN1 is significantly lower in the BECN1 KO MCF-7 cell lines compared to the wild-type MCF-7 cell lines. Hence, the knockdown of endogenous BECN1protein expression in MCF-7 KO cells is correlated with a loss of BECN1 antibody (MBS2028535) staining. This loss of the BECN1 WB band identified in the wild-type MCF-7 cells is a powerful validation of the specificity of the MBS2028535 BECN1 antibody for BECN1. KO antibody validation is among the most highly types of specificity validations that an antibody can undergo and is highly sought after by the research community.