User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/09/26: 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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* The end of pump A was inserted to the bottle solution of binding buffer; pump B was inserted into the bottle of the elution buffer. The pump was set at pump wash basic. | * The end of pump A was inserted to the bottle solution of binding buffer; pump B was inserted into the bottle of the elution buffer. The pump was set at pump wash basic. | ||
* In consecutive order, the column was flushed with 25 mL of the binding buffer, 25 mL of elution buffer, and another 25 mL of binding buffer. | * In consecutive order, the column was flushed with 25 mL of the binding buffer, 25 mL of elution buffer, and another 25 mL of binding buffer. | ||
* The flowpath was switched to position 3 after the run through of buffers. The pump flow was at 0.15 MPa and the flowrate was set to 5 mL/min. | * The flowpath was switched to position 3 after the run through of buffers. The pump flow was at 0.15 MPa and the flowrate was set to 5 mL/min. | ||
* From the readings, the detection indicates that the absorbance is at 280; indicative of the presence of Histidine. However, the absorbance at 280 is not exclusive to Histidine alone but also corresponds to Tryptophan, Tyrosine, and Phenylalanine. | |||
* The conductivity reading is sensitive to the amount of salt in the buffer. | |||
* On the dialog box, pump > gradient > B > 100%. | |||
* Once the peak levels back to baseline, pump > gradient > B > 0%. This washes the system with A. | |||
The conductivity | * At Flowpath > injection valve > position > inject. Switch to position 1. This bypasses the column with the superloop's binding buffer flow at at 10 mL/min on inject. Then press end. | ||
* Sample 1 was loaded onto the super loop with the syringe at inject. The position was switched at position 3. The flow rate was at 5 mL/min with the gradient of B at 0%. From inject, the setting was switch to load. | |||
* The desired baseline should reach an absorbance of 280 and 250. Then hit pause. | |||
The | * The fraction is under the Frac 900 function in the dialog box. 5 mL fraction size was chosen. Then hit continue. | ||
* On the pump function, the load was set at 0% B after 25 mL the flow rate of 0 mL/min. was changed to 5 mL/min. | |||
The | * The same steps were done for sample 2. | ||
The | * The filtrate for sample 1 was collected from tubes 1-6 and sample 2 was collected from tubes 7-11. | ||
* Observing the graph below, the peak at fraction 2 indicates that most of the protein (ADA) was collected in this run. For sample 2, the protein filtrate was mostly collected at fraction 8. | |||
[[Image:ADA HisTrapE.PNG]] | |||
==Preparation of Au/BSA solution== | |||
* The nanoparticles were synthesized from 15 μM BSA with 2 mL of .0336 M Au diluted down to 10 mL of water. | |||
* The solutions were prepared with the same standard mole ratios as before. | |||
* Then, the solutions were placed to the thermo scientific for 4 h. at 80°C. | |||
Latest revision as of 22:06, 26 September 2017
Project name | Main project page Previous entry Next entry |
Purification of Proteins
Preparation of Au/BSA solution
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