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=BSA Ab-Oligo Linkage-Version 3 Probes (Started [[Daniel:Notebook/ComboLock/2017-5-13|Saturday May 13]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] Checking concentration and preparing for a BSA experiment tomorrow. To check concentration I'll use the Bradford assay. ==Protocol== <ol> <li>Bradford Assay (BioRad)</li> <ol type="A"> <li>Prepare dye reagent by diluting 200 uL Dye Reagent Concentrate with 800 uL (1:5 dilution) ddH<sub>2</sub>O</li> <li>Filter using whatman #1 paper or similar (15mm diameter circles)</li> <li>Prepare three to five dilutions of a protein standard (use BSA)</li> <ol type="a"><li>The linear range of the assay for BSA is 0.2 to 0.9 mg/ml</li></ol> <li>Pipet 100 uL each standard and sample solution into a clean test tube</li> <li>Add 5.0 mL diluted dye reagent to each tube and vortex</li> <li>Incubate at RT for >5 min but <1 hr</li> <li>Measure absorbance at 595 nm</li> </ol></ol> <ol> <li>Bradford Assay (BioRad)</li> <ol type="A"> <li>Prepare dye reagent by diluting 200 uL Dye Reagent Concentrate with 800 uL (1:5 dilution) ddH<sub>2</sub>O</li> <li>Filter using whatman #1 paper or similar (15mm diameter circles)</li> <li>Prepare four dilutions of BSA-biotin from stock (2 mg/mL undiluted; BSA linear range:0.9-0.2 mg/mL)</li> <ol type="a"><li>'''S1''':2 uL BSA0, 2 uL PBS (1 mg/mL)</li> <li>'''S2''':1 uL BSA0, 3 uL PBS (0.5 mg/mL)</li> <li>'''S3''':1 uL BSA0, 5 uL PBS (0.33 mg/mL)</li> <li>'''S4''':4 uL BSA1 (0.2 mg/mL)</li></ol> <li>Mix 196 uL diluted reagent and 4 uL sample into a 0.5mL tube</li> <li>Incubate at RT for 5 min</li> <li>Measure absorbance at 595 nm</li> </ol></ol> ==Results== [[Image:BradfordCurve-20170613.png|440x325px]] {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#B1A0C7;font-size:12pt" | align="center" width="65" height="46" valign="bottom" | |style="font-weight:bold" width="65" align="center" | Sample Reading |style="font-weight:bold" width="122" align="center" | Approx. Concentration (ug/mL) |style="font-weight:bold" width="99" align="center" | Approximate final volume |style="font-weight:bold" width="65" align="center" | uL EDTA (500 mM) |style="font-weight:bold" width="81" align="center" | Salmon Sperm DNA (1 mg/mL) |style="font-weight:bold" width="84" align="center" | uL fish gelatin (5%) |style="font-weight:bold" width="65" align="center" | uL Tris HCl (500 mM) |style="font-weight:bold" width="65" align="center" | 1X PBS |- style="font-size:12pt" | height="15" valign="bottom" | Ab-A | align="center" align="center" valign="bottom" | 0.008 | align="center" align="center" valign="bottom" | 155 | align="center" align="center" valign="bottom" | 40 | align="center" align="center" valign="bottom" | 0.32 | align="center" align="center" valign="bottom" | 1.4 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 1.6 | align="center" align="center" valign="bottom" | 15 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Ab-B | align="center" align="center" valign="bottom" | 0.01 | align="center" align="center" valign="bottom" | 250 | align="center" align="center" valign="bottom" | 70 | align="center" align="center" valign="bottom" | 0.56 | align="center" align="center" valign="bottom" | 2.45 | align="center" align="center" valign="bottom" | 3.5 | align="center" align="center" valign="bottom" | 2.8 | align="center" align="center" valign="bottom" | 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 [[Category:ComboLock]] [[Category:20170513]]
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