Daniel:Notebook/ComboLock/2017-6-13
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BSA Ab-Oligo Linkage-Version 3 Probes (Started Saturday May 13)[edit]
Checking concentration and preparing for a BSA experiment tomorrow. To check concentration I'll use the Bradford assay.
Protocol[edit]
- Bradford Assay (BioRad)
- Prepare dye reagent by diluting 200 uL Dye Reagent Concentrate with 800 uL (1:5 dilution) ddH2O
- Filter using whatman #1 paper or similar (15mm diameter circles)
- Prepare three to five dilutions of a protein standard (use BSA)
- The linear range of the assay for BSA is 0.2 to 0.9 mg/ml
- Pipet 100 uL each standard and sample solution into a clean test tube
- Add 5.0 mL diluted dye reagent to each tube and vortex
- Incubate at RT for >5 min but <1 hr
- Measure absorbance at 595 nm
- Bradford Assay (BioRad)
- Prepare dye reagent by diluting 200 uL Dye Reagent Concentrate with 800 uL (1:5 dilution) ddH2O
- Filter using whatman #1 paper or similar (15mm diameter circles)
- Prepare four dilutions of BSA-biotin from stock (2 mg/mL undiluted; BSA linear range:0.9-0.2 mg/mL)
- S1:2 uL BSA0, 2 uL PBS (1 mg/mL)
- S2:1 uL BSA0, 3 uL PBS (0.5 mg/mL)
- S3:1 uL BSA0, 5 uL PBS (0.33 mg/mL)
- S4:4 uL BSA1 (0.2 mg/mL)
- Mix 196 uL diluted reagent and 4 uL sample into a 0.5mL tube
- Incubate at RT for 5 min
- 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