For streptavidin blotting, HEK293T cells in wells of a 6-well plate were transfected with APXNES variants and labeled under the same conditions described for biotin-phenol imaging above. After 1 minute of labeling, the cells were washed 3 times with quencher solution (10 mM sodium azide, 10 mM sodium ascorbate, and 5 mM Trolox), then scraped and pelleted by centrifugation at 200 rpm for 5 minutes. The pellet was stored at -80 °C, then lysed with RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 1 × protease cocktail (Sigma Aldrich, catalog no. P8849), 1 mM PMSF (phenylmethylsulfonyl fluoride), 10 mM sodium azide, 10 mM sodium ascorbate, and 5 mM Trolox) for 1 minute at 4 °C. The cell pellet was resuspended by gentle pipetting. Lysates were clarified by centrifugation at 13,000 rpm for 10 minutes at 4 °C before separation on a 9% SDS-PAGE gel. For blotting analysis, gels were transferred to nitrocellulose membrane, stained by Ponceau S (10 minutes in 0.1% w/v Ponceau S in 5% acetic acid/water), and blocked with “blot blocking buffer” (3% w/v BSA and 0.1% Tween-20 in Tris-buffered saline) at 4 °C overnight. The blots were immersed in streptavidin-HRP in blot blocking buffer (1:3000 dilution, Thermo Scientific) at room temperature for 60 minutes, then rinsed with blot blocking buffer 5 × 5 minutes before development with Clarity™ reagent (Bio-Rad) and imaging on an Alpha Innotech gel imaging system. For assessing comparative enzyme expression level, an identical gel and blot was prepared in parallel, and immunoblotted with α-FLAG-M2-HRP (1:3000, Sigma).
For blotting of endogenous proteins, HEK293T cells were grown in 6-well plates and infected at ∼50% confluency with 500 μL lentivirus prepared as described above. After 48 hours, cells were split into T25 flasks. From the same suspensions, cells were also plated into 48-well plates for side-by-side biotinphenol and immunofluorescence to check for enzyme activity and comparable enzyme expression levels. Biotin phenol labeling in the T25 flasks was performed with 30 minutes preincubation of 500 μM biotinphenol in 4 mL of a 1:1 mixture of DMEM:MEM (both from Cellgro) at 37°C. Flasks were quickly inverted so biotin-phenol media was on the ceiling of the flask, and 40 μL of 100 mM H2O2 was added (for a final concentration of 1 mM) to the media and agitated to mix. Flasks were again inverted and cells were exposed to the 1 mM H2O2 and 500 μM biotin-phenol solution for 1 minute at room temperature. After the 1 minute duration, the flask was inverted and the solution replaced with 5 mL of ice cold DPBS with quenchers (5 mM Trolox, 10 mM sodium ascorbate, 10 mM sodium azide) and the flask re-inverted for 1 minute. The flask was then washed with 5 mL DPBS + quenchers two more times. Cells were then resuspended by electronic pipette in 5 mL DPBS + quenchers and pelleted at 3000g for 10 minutes at 4°C. The supernatant was removed and the pellet was frozen at -80°C overnight.
Enrichment of endogenous biotinylated proteins was performed according to previous protocols. Cell pellets were lysed in 600 μL RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, pH 7.5), with 1 × protease cocktail (Sigma Aldrich, catalog no. P8849), 1 mM PMSF (phenylmethylsulfonyl fluoride), 5 mM Trolox, 10 mM sodium ascorbate, and 10 mM sodium azide. The lysates were centrifuged at 15,000g for 10 minutes at 4°C and the supernatant was transferred to a new 1.5 mL microcentrifuge tube as whole cell lysate (WCL). Protein concentrations of each WCL were determined by the Pierce 660 nm Protein Assay (catalog no. 22660).
Streptavidin-coated magnetic beads (Pierce, catalog no. 88817) were washed twice with RIPA buffer and 1750 μg of each WCL sample was incubated with 120 μL of magnetic bead slurry in separate 1.5 mL microcentrifuge tubes with rotation for 1 hour at room temperature. The beads were subsequently washed twice with 1 mL RIPA lysis buffer, once with 1 mL of 1 M KCl in ddH2O, once with 1 mL of 0.1M Na2CO3 in ddH2O, once with 1 mL of 2 M urea in 10 mM Tris-HCl pH 8.0, and twice with 1 mL RIPA lysis buffer. Biotinylated proteins were eluted by boiling the beads in 75 μl 3 × protein loading buffer supplemented with 20 mM dithiothreitol (DTT) and 2 mM free biotin. 17.5 μg of each WCL and 25 μl of each streptavidin eluate (SAE) were separated on 9% SDS-PAGE gels.
Nitrocellulose blot transfer and Ponceau S staining were performed as described above. Membranes were blocked with blot blocking buffer overnight at 4°C, cut into strips based on MW, and incubated facedown into 500 μl of blot blocking buffer containing the specified primary antibodies (see table below) for 1 hour at room temperature. Blots were washed 5 × 5 minutes in blot blocking buffer before incubation in 10 mL of secondary antibody in blot blocking buffer for 1 hour at room temperature. The membranes were then washed again 5 × 5 minutes in blot blocking buffer before imaging with Clarity™ reagent (Bio-Rad) as described above.
For blotting of endogenous proteins, HEK293T cells were grown in 6-well plates and infected at ∼50% confluency with 500 μL lentivirus prepared as described above. After 48 hours, cells were split into T25 flasks. From the same suspensions, cells were also plated into 48-well plates for side-by-side biotinphenol and immunofluorescence to check for enzyme activity and comparable enzyme expression levels. Biotin phenol labeling in the T25 flasks was performed with 30 minutes preincubation of 500 μM biotinphenol in 4 mL of a 1:1 mixture of DMEM:MEM (both from Cellgro) at 37°C. Flasks were quickly inverted so biotin-phenol media was on the ceiling of the flask, and 40 μL of 100 mM H2O2 was added (for a final concentration of 1 mM) to the media and agitated to mix. Flasks were again inverted and cells were exposed to the 1 mM H2O2 and 500 μM biotin-phenol solution for 1 minute at room temperature. After the 1 minute duration, the flask was inverted and the solution replaced with 5 mL of ice cold DPBS with quenchers (5 mM Trolox, 10 mM sodium ascorbate, 10 mM sodium azide) and the flask re-inverted for 1 minute. The flask was then washed with 5 mL DPBS + quenchers two more times. Cells were then resuspended by electronic pipette in 5 mL DPBS + quenchers and pelleted at 3000g for 10 minutes at 4°C. The supernatant was removed and the pellet was frozen at -80°C overnight.
Enrichment of endogenous biotinylated proteins was performed according to previous protocols. Cell pellets were lysed in 600 μL RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, pH 7.5), with 1 × protease cocktail (Sigma Aldrich, catalog no. P8849), 1 mM PMSF (phenylmethylsulfonyl fluoride), 5 mM Trolox, 10 mM sodium ascorbate, and 10 mM sodium azide. The lysates were centrifuged at 15,000g for 10 minutes at 4°C and the supernatant was transferred to a new 1.5 mL microcentrifuge tube as whole cell lysate (WCL). Protein concentrations of each WCL were determined by the Pierce 660 nm Protein Assay (catalog no. 22660).
Streptavidin-coated magnetic beads (Pierce, catalog no. 88817) were washed twice with RIPA buffer and 1750 μg of each WCL sample was incubated with 120 μL of magnetic bead slurry in separate 1.5 mL microcentrifuge tubes with rotation for 1 hour at room temperature. The beads were subsequently washed twice with 1 mL RIPA lysis buffer, once with 1 mL of 1 M KCl in ddH2O, once with 1 mL of 0.1M Na2CO3 in ddH2O, once with 1 mL of 2 M urea in 10 mM Tris-HCl pH 8.0, and twice with 1 mL RIPA lysis buffer. Biotinylated proteins were eluted by boiling the beads in 75 μl 3 × protein loading buffer supplemented with 20 mM dithiothreitol (DTT) and 2 mM free biotin. 17.5 μg of each WCL and 25 μl of each streptavidin eluate (SAE) were separated on 9% SDS-PAGE gels.
Nitrocellulose blot transfer and Ponceau S staining were performed as described above. Membranes were blocked with blot blocking buffer overnight at 4°C, cut into strips based on MW, and incubated facedown into 500 μl of blot blocking buffer containing the specified primary antibodies (see table below) for 1 hour at room temperature. Blots were washed 5 × 5 minutes in blot blocking buffer before incubation in 10 mL of secondary antibody in blot blocking buffer for 1 hour at room temperature. The membranes were then washed again 5 × 5 minutes in blot blocking buffer before imaging with Clarity™ reagent (Bio-Rad) as described above.
