User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/10/14: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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* Inserted screws to prevent evaporation | * Inserted screws to prevent evaporation | ||
* Placed on low speed shaker for 1 week | * Placed on low speed shaker for 1 week | ||
==Dialysis Data== | |||
<u>Bradford Analysis</u> | |||
*Only running bradford analysis of protein-containing solutions, that is Colloid 1, 2, 3, 4 and 5, since the protein cannot diffuse through 3500 MWCO. | |||
* Remove 100 μL of solution from each chamber (7 in all) and run Bradford analysis | |||
**Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl | |||
**Recall, 100 μL solution + 200 μL diluted Bradford + 700 μL Tris/NaCl buffer | |||
**PS cuvettes, measuring 400 - 800 nm | |||
[[Image:Bradford Colloid 141014.png]] | |||
<u>Bradford Calibration curve</u> | |||
*Did Bradford analysis for undialysed lysozyme solutions with concentrations 0.12, 0.3, 0.6 and 1 g/L. | |||
*The calibration curve below: | |||
[[Image:Lysozyme-Bradford CV.png]]<br.> | |||
<u>UV-Vis Absorption</u> <br.> | |||
Transfer 50 μL to a small volume UV cuvette & measure UV absorption | |||
**Be sure your cuvette is clean before hand. Use SDS, HCl, HPLC, & methanol washes | |||
**Measure entire 200 - 400 nm range. (Care about peak at 280 nm. Need to compare to fluorescence data to see how the binding is affected) | |||
<u>Fluorescence</u> | |||
*Measured fluorescence of dyalised solutions | |||
*Fluorescence (glass microcuvette & 100 fold diluted) for all protein solutions | |||
**10μL of lysozyme were diluted to 1mL by adding HPLC. (Remember to account for dilution factor later on) | |||
<u>Ca2+ ISE</u> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''CaCl<sub>2</sub> concentration''' | |||
| align="center" style="background:#f0f0f0;"|'''mV measurement (mV)''' | |||
|- | |||
| 5μM || 31.8 | |||
|- | |||
| 50μM || 40.6 | |||
|- | |||
| 500μM || 40.1 | |||
|- | |||
| 5mM || 56.6 | |||
|- | |||
| 50mM || 78.5 | |||
|- | |||
|} | |||
===Buffer Preparation=== | ===Buffer Preparation=== | ||
* | * Prepared 50 mL 66 mM potassium phosphate buffer, pH 6.319 | ||
**Use HPLC water | **Use HPLC water | ||
**8.98 mg/mL KH<sub>2</sub>PO<sub>4</sub> should produce pH 4 - 4.1 | **8.98 mg/mL KH<sub>2</sub>PO<sub>4</sub> should produce pH 4 - 4.1 | ||
**Add 1 M KOH to adjust pH to 6.24. '''To avoid this step a mixture of monobasic and dibasic potassium phosphate was used'''. | **Add 1 M KOH to adjust pH to 6.24. '''To avoid this step a mixture of monobasic and dibasic potassium phosphate was used'''. | ||
***0.00265 mol × 136.09 g/mol = 0.361 g monobasic KH<sub>2</sub>PO<sub>4</sub> | ***0.00265 mol × 136.09 g/mol = 0.361 g monobasic KH<sub>2</sub>PO<sub>4</sub> | ||
***0.0006 mol × 174.18g/mol = 0.105 g dibasic | ***0.0006 mol × 174.18g/mol = 0.105 g dibasic KH<sub>2</sub>PO<sub>4</sub> | ||
**Store | **Store | ||
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Latest revision as of 00:26, 27 September 2017
Project name | Main project page Previous entry Next entry | ||||||||||||
Tasks for October 14
SDS-PAGE #2Procedures as listed by Dr. Fox on Oct. 14
Preparation of 30:1 colloid solution vs CaCl2 using MWCO 3500 dialysisA new dialysis experiment was prepared using 3,500 MWCO tubing. <br.> The wells were matched up in the following way. Note that 1 mL of the following were added to each well:
Dialysis DataBradford Analysis
Bradford Calibration curve
<br.> UV-Vis Absorption <br.> Transfer 50 μL to a small volume UV cuvette & measure UV absorption
Fluorescence
Ca2+ ISE
Buffer Preparation
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