Daniel:Notebook/ComboLock/2017-6-13

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BSA Ab-Oligo Linkage-Version 3 Probes (Started Saturday May 13)[edit]

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Checking concentration and preparing for a BSA experiment tomorrow. To check concentration I'll use the Bradford assay.

Protocol[edit]

  1. Bradford Assay (BioRad)
    1. Prepare dye reagent by diluting 200 uL Dye Reagent Concentrate with 800 uL (1:5 dilution) ddH2O
    2. Filter using whatman #1 paper or similar (15mm diameter circles)
    3. Prepare three to five dilutions of a protein standard (use BSA)
      1. The linear range of the assay for BSA is 0.2 to 0.9 mg/ml
    4. Pipet 100 uL each standard and sample solution into a clean test tube
    5. Add 5.0 mL diluted dye reagent to each tube and vortex
    6. Incubate at RT for >5 min but <1 hr
    7. Measure absorbance at 595 nm
  1. Bradford Assay (BioRad)
    1. Prepare dye reagent by diluting 200 uL Dye Reagent Concentrate with 800 uL (1:5 dilution) ddH2O
    2. Filter using whatman #1 paper or similar (15mm diameter circles)
    3. Prepare four dilutions of BSA-biotin from stock (2 mg/mL undiluted; BSA linear range:0.9-0.2 mg/mL)
      1. S1:2 uL BSA0, 2 uL PBS (1 mg/mL)
      2. S2:1 uL BSA0, 3 uL PBS (0.5 mg/mL)
      3. S3:1 uL BSA0, 5 uL PBS (0.33 mg/mL)
      4. S4:4 uL BSA1 (0.2 mg/mL)
    4. Mix 196 uL diluted reagent and 4 uL sample into a 0.5mL tube
    5. Incubate at RT for 5 min
    6. Measure absorbance at 595 nm

Results[edit]

File:BradfordCurve-20170613.png

  Sample Reading Approx. Concentration (ug/mL) Approximate final volume uL EDTA (500 mM) Salmon Sperm DNA (1 mg/mL) uL fish gelatin (5%) uL Tris HCl (500 mM) 1X PBS
Ab-A 0.008 155 40 0.32 1.4 2 1.6 15
Ab-B 0.01 250 70 0.56 2.45 3.5 2.8 41

Final Composition:

  • 75 ug/mL (~0.5 uM) antibody
  • 35 ug/mL salmon sperm DNA
  • 4 mM EDTA
  • 20 mM Tris-HCl
  • 0.1% fish gelatin
  • 1X PBS