Human BAX constructs (wild-type, C126A) were cloned into the pMIG (MSCV-IRES-GFP) vector, and the presence of insert and the indicated mutation were confirmed by DNA sequencing. Transfection of the packaging cell line GPG-293 yielded amphotropic retroviral particles, which were collected by filtration and ultracentrifugation, and then used to reconstitute Bax−/−Bak−/− MEFs with the indicated BAX proteins, as described (Gavathiotis et al., 2008 (link); Kim et al., 2006 (link)). The reconstituted MEFs were sorted for GFP-positivity over two rounds of flow cytometry to ensure comparable levels of expression. Transduction was verified by anti-BAX western analysis using the 2D2 antibody (Santa Cruz).
2d2 antibody
Manufactured by Santa Cruz Biotechnology
The 2D2 antibody is a monoclonal antibody produced by Santa Cruz Biotechnology. It is designed for use in various laboratory applications, such as Western blotting, immunoprecipitation, and immunohistochemistry. The core function of the 2D2 antibody is to specifically recognize and bind to its target antigen.
Automatically generated - may contain errors
4 protocols using 2d2 antibody
1
Retroviral reconstitution of Bax-deficient cells
2
Reconstitution of Bax Mutants in Bax/Bak Deficient MEFs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human BAX constructs (wild-type, L113A, F114A, Y115A, and F116A) were cloned (Genewiz) into the pMIG II (pMSCV-IRES-GFP II) vector (Addgene #52107), and the presence of insert and the indicated mutations were confirmed by DNA sequencing. Transfection of the packaging cell line GPG-293 yielded amphotropic retroviral particles, which were collected by filtration and precipitation with PEG-it solution (System Biosciences), and then used to reconstitute Bax−/−Bak−/− MEFs with the indicated BAX mutants, as described27 (link),44 (link). The reconstituted MEFs were sorted for GFP-positivity over two rounds of flow cytometry to ensure comparable levels of expression. Transduction was verified by BAX western analysis using the 2D2 antibody (Santa Cruz Biotechnology Cat# sc-20067; RRID: AB_626726; 1:200). Cells were maintained in Dulbecco’s Modified Eagle Medium (GIBCO) with 10% FBS, 100 U/mL penicillin and streptomycin, and 2 mM glutamine. Cells were verified as mycoplasma-negative using the MycoAlert mycoplasma detection kit (Lonza Biologics).
3
Retroviral reconstitution of Bax-deficient cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human BAX constructs (wild-type, C126A) were cloned into the pMIG (MSCV-IRES-GFP) vector, and the presence of insert and the indicated mutation were confirmed by DNA sequencing. Transfection of the packaging cell line GPG-293 yielded amphotropic retroviral particles, which were collected by filtration and ultracentrifugation, and then used to reconstitute Bax−/−Bak−/− MEFs with the indicated BAX proteins, as described (Gavathiotis et al., 2008 (link); Kim et al., 2006 (link)). The reconstituted MEFs were sorted for GFP-positivity over two rounds of flow cytometry to ensure comparable levels of expression. Transduction was verified by anti-BAX western analysis using the 2D2 antibody (Santa Cruz).
4
Mitochondrial Fractionation and Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bax−/−Bak−/− MEFs and those reconstituted with the indicated BAX proteins were cultured as described above and 2 × 107 cells were harvested for fractionation. Cell pellets were resuspended in isolation kit buffers (ThermoFisher) and lysed by Dounce homogenization. Mitochondrial and cytosolic fractions were purified by differential centrifugation, performed in accordance with the manufacturer’s protocol. Mitochondria were lysed in mitochondrial lysis buffer (25 mM Tris, 150 mM NaCl, 2% CHAPS, pH 7.2) and protein quantitation of the supernatant and cytosolic fractions was performed using the BCA assay (ThermoFisher). Samples were subjected to electrophoresis and western blotting using the 2D2 antibody (Santa Cruz Biotechnology Cat# sc-20067; RRID: AB_626726; 1:200). Western analysis using VDAC1 (Abcam cat #ab186321; 1:1000), LDH (Abcam cat #ab47010, RRID:AB_1952042; 1:1000), and actin (Cell Signaling Technology Cat# 5125, RRID:AB_1903890; 1:1000) antibodies was performed to verify mitochondrial and cytosolic fractions, and equal protein loading, respectively.
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!