biotinylated cell surface proteins  (Vector Laboratories)

Journal: Biomolecules

Article Title: CD45 and Basigin (CD147) Are Functional Ligands for Galectin-8 on Human Leukocytes

doi: 10.3390/biom15091243

Figure Lengend Snippet: Validation of Gal-8 chromatography columns. ( A ) Schematic representation of Gal-8 chromatography with HL-60 cell membrane preparations and steps in the procedure. Gal-8 chromatography (1 mg Gal-8/mL beads) with ( B ), positive control: mouse laminin (LA); ( C ) negative control: CHO-Lec8 cell mutant membrane (250 μg/mL); ( D ) HL-60 cell membrane extract (250 μg/mL). ( B – D ) were visualized with silver nitrate staining. ( E , F ) Blotting of fractions from ( D ) with biotinylated Gal-8 (1 μg/mL) in absence ( E ) and presence ( F ) of lactose. Legend: MW, molecular weight markers; LA, laminin; MB, total membrane fraction; FT, flow through; W, wash; S, 100 mM sucrose wash; L, 100 mM lactose elution.

Article Snippet: After SDS-PAGE and transferring the blot with Transblot (Bio-Rad, Hercules, CA, USA), biotinylated cell surface proteins were detected by Streptavidin-horseradish peroxidase (Strep-HRP, Vector Labs, Newark, CA, USA) at a 1:30,000 dilution in Tris buffer saline (TBS) containing 0.1% Tween-5% bovine serum albumin (TBST-BSA) after blocking overnight at 4 °C or 1 h at room temperature (RT) in TBST-BSA.

Techniques: Biomarker Discovery, Chromatography, Membrane, Positive Control, Negative Control, Mutagenesis, Staining, Molecular Weight

Journal: Biomolecules

Article Title: CD45 and Basigin (CD147) Are Functional Ligands for Galectin-8 on Human Leukocytes

doi: 10.3390/biom15091243

Figure Lengend Snippet: Gal-8 chromatography with intact HL-60 cells. ( A ) Schematic representation of Gal-8 chromatography steps with intact HL-60 cells. ( B ) Gal-8 chromatography (1 mg Gal-8/mL beads) of intact, cell surface biotinylated HL-60 cells. Samples were visualized with silver nitrate staining. ( C ) Strep-HRP blot (1 μg/mL) of fractions from B with intact biotinylated HL-60 cells. Legend: MW, molecular weight markers; MB, total membrane fraction; FT, flow through; W, wash; S, 100 mM sucrose wash; L, 100 mM lactose elution.

Article Snippet: After SDS-PAGE and transferring the blot with Transblot (Bio-Rad, Hercules, CA, USA), biotinylated cell surface proteins were detected by Streptavidin-horseradish peroxidase (Strep-HRP, Vector Labs, Newark, CA, USA) at a 1:30,000 dilution in Tris buffer saline (TBS) containing 0.1% Tween-5% bovine serum albumin (TBST-BSA) after blocking overnight at 4 °C or 1 h at room temperature (RT) in TBST-BSA.

Techniques: Chromatography, Staining, Molecular Weight, Membrane

Journal: Biomolecules

Article Title: CD45 and Basigin (CD147) Are Functional Ligands for Galectin-8 on Human Leukocytes

doi: 10.3390/biom15091243

Figure Lengend Snippet: CD45 was detected among the Gal-8 binding proteins on HL-60 cells. ( A ) Anti-CD45 blot of full-length Gal-8 chromatography with HL-60 cell membranes; ( B ) anti-CD45 blot of Gal-8C chromatography with HL-60 cell membranes; ( C ) Reverse Transcriptase-PCR of CD45 isoforms and beta-actin with HL-60 and Jurkat cells. Specific amplicons for CD45 (about 200, 350, and 500 bp) are visible; ( D ) HL-60 cell staining with anti-CD45-Alexa 488 or isotype control measured by flow cytometry; ( E ) confocal images of HL-60 cells treated at 4 °C with biotinylated Gal-8CM ( top row ) or Gal-8NM ( bottom row ) with Strep-Alexa 633 (red) and anti-CD45-Alexa 488 (green); Merge 1: whole cell reconstruction, Merge 2: detailed colocalization (yellow), scale bar = 5 μm. White arrows indicate colocalization in punctate membrane microdomains. Legend: MB, total membrane fraction; FT, flow through; W, wash; S, 100 mM sucrose wash; L, 100 mM lactose elution; MW, molecular weight.

Article Snippet: After SDS-PAGE and transferring the blot with Transblot (Bio-Rad, Hercules, CA, USA), biotinylated cell surface proteins were detected by Streptavidin-horseradish peroxidase (Strep-HRP, Vector Labs, Newark, CA, USA) at a 1:30,000 dilution in Tris buffer saline (TBS) containing 0.1% Tween-5% bovine serum albumin (TBST-BSA) after blocking overnight at 4 °C or 1 h at room temperature (RT) in TBST-BSA.

Techniques: Binding Assay, Chromatography, Reverse Transcription, Staining, Control, Flow Cytometry, Membrane, Molecular Weight

Journal: Biomolecules

Article Title: CD45 and Basigin (CD147) Are Functional Ligands for Galectin-8 on Human Leukocytes

doi: 10.3390/biom15091243

Figure Lengend Snippet: Both N- and C-terminal domains of Gal-8 bind to basigin and colocalize with basigin on HL-60 cells. ( A ) Anti-basigin (CD147) blot of Gal-8NM and Gal-8CM chromatography with HL-60 cell membranes (U: Unbound, B: Bound); ( B ) HL-60 cell staining with anti-basigin-Alexa 488 or isotype control measured by flow cytometry; ( C ) confocal images of HL-60 cells treated at 4 °C with (a) biotinylated Gal-8NM or (e) Gal-8CM with Strep-Alexa 633 (red) and (b,f) anti-basigin-Alexa 488 (green); Merged images Gal-8NM/anti-basigin and Gal-8CM/anti-basigin are shown (c,g), and zoomed images on two individual cells from (c,g) are shown (d,h), respectively. Scale bar represents 5 μm.

Article Snippet: After SDS-PAGE and transferring the blot with Transblot (Bio-Rad, Hercules, CA, USA), biotinylated cell surface proteins were detected by Streptavidin-horseradish peroxidase (Strep-HRP, Vector Labs, Newark, CA, USA) at a 1:30,000 dilution in Tris buffer saline (TBS) containing 0.1% Tween-5% bovine serum albumin (TBST-BSA) after blocking overnight at 4 °C or 1 h at room temperature (RT) in TBST-BSA.

Techniques: Chromatography, Staining, Control, Flow Cytometry