Description
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Comparison of elution strategies for HiBiT-tagged firefly luciferase immunoprecipitated from HEK293 cells. Starting samples consisted of lysate containing 5 × 10 6 cells/ml in Mammalian Lysis Buffer (Cat.# G9381) supplemented with Protease Inhibitor Cocktail (Cat.# G6521) and RQ1 RNase-Free DNase (Cat.# M6101). Cleared lysate was incubated with beads for 2 hours at 4°C. Eluates were generated using four different elution conditions: 1) SDS loading buffer; 2) low-pH buffer; 3) DrkBiT Elution Peptide (Cat.# N7400, diluted 100-fold in TBST) incubated for 2 hours at room temperature; or 4) DrkBiT Elution Peptide incubated overnight at 4°C. TBST-only buffer controls were performed for comparison to DrkBiT Peptide eluates. Eluates were generated using a 4-fold lower volume (100µl) than the starting lysate samples (400µl). Equivalent volumes of the starting lysate sample, supernatants and eluates were used for analysis. Panel A shows analysis of samples using the Nano-Glo® HiBiT Blotting System (Cat.# N2410). The left gel photo is scaled to lower intensities to illustrate effective clearance of Fluc-HiBiT from supernatants by Anti-HiBiT Magne® Beads but not control beads without antibody. The right gel photo is scaled to higher intensities to demonstrate increased concentrations of Fluc-HiBiT in the eluates compared to the starting sample. In Panel B , the enzymatic activity of firefly luciferase was measured using the ONE-Glo™ Assay (Cat.# E6110), highlighting the ability to gently elute HiBiT-tagged proteins and maintain enzyme activity using the DrkiBiT Elution Peptide.
CRISPR-mediated HiBiT-tagging enables immunoprecipitation of proteins at endogenous levels. Using CRISPR/Cas9 genome-editing technology, the HiBiT tag was added to termini of BRD2, PARP1 and BTK in HEK293, DLD1 and K562 cells, respectively. The standard protocol in Section 3.B of Technical Manual #TM771 was used to immunoprecipitate the proteins. Samples were analyzed to monitor protein clearance from the supernatant and protein recovery in the eluate. Starting samples of cleared cell lysate (1ml; 5 × 10 6 cells/ml) were immunoprecipitated by binding overnight at 4°C and eluting in 80µl of 0.1M glycine (pH 2.5). Eluates were removed from beads and neutralized with 20µl of 2M Tris (pH 7.5). Equivalent volumes of each starting lysate sample, supernatant and eluate were analyzed using the Nano-Glo® HiBiT Blotting System (Cat.# N2410).
HiBiT-based IP-MS identifies SMARCA2-associated SWI/SNF proteins. The HiBiT tag was inserted at the C-terminus of endogenous SMARCA2 in HeLa cells using CRISPR/Cas9 genome editing. Triplicate immunoprecipitations were performed from lysates of both edited and parental cells using the standard protocol described in Section 3.B of Technical Manual #TM771. Eluates were processed with the Magnetic Proteomics Sample Prep Kit (Cat.# CS3325A04) and digested with Trypsin/Lys-C (Cat.# V5071). Peptides were analyzed by LC-MS/MS on a Thermo Fisher Scientific Orbitrap Exploris 240. Data were searched using ProteomeDiscoverer 3.1 against the Uniprot Human database using the Sequest search algorithm. Red circles in the volcano plot highlight known SMARCA2 interactors, including members of the SWI/SNF chromatin remodeling complex.
