User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/09/26

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(Purification of Proteins)
Current revision (09:49, 7 December 2012) (view source)
(Purification of Proteins)
 
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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%.
-
We needed to run 25mL of Buffer through the column at a pressure of .14/.15 ( the pressure tells if there is anything clogging the system)
+
* Once the peak levels back to baseline, pump > gradient > B > 0%. This washes the system with A.
-
The conductivity meter reads how much salt is in the system
+
* 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.
-
Imidizole also contains the functional group of histidine.
+
* 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.
-
We needed to first run the binding buffer, followed by the elution buffer, and then the binding buffer once more through the column otherwise the column will have too much imidazole.
+
* The desired baseline should reach an absorbance of 280 and 250. Then hit pause.
-
The column only run at 5mL/minute. The nickel column of the instrument can hold 5mL and contains polymer beads. The ADA protein contains specifically a histag on the protein of 6 histidines (which is the amount necessary in order to have a night enough affinity to nickel). Histidine has an affinity to nickel and therefore will bind with it.
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* The fraction is under the Frac 900 function in the dialog box. 5 mL fraction size was chosen. Then hit continue.
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We then put the protein on. We ran sample 1 first.
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* 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.
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The protein histidines (in the ADA) bind with the nickel in the column. To get the protein out, we use the elution buffer because the imidazole histidines compete with the histidine in the column and binds to the nickel. This results in the protein being pushed out.
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* The same steps were done for sample 2.
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The purified protein was then collected in different Fracs (test tubes each containing 5mL) For sample 1, most of the protein was in Frac #2 ( as was indicated by the detector on the FPLC) and for the other full sample, most of the protein was collected in frac #8. Regardless, all the fracs were collected, labelled and stored in the refrigerator.
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* The filtrate for sample 1 was collected from tubes 1-6 and sample 2 was collected from tubes 7-11.
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* 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.

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Purification of Proteins

  • Purification was achieved by running the collected filtrate through an affinity chromatography that is attached to a Fast Protein Liquid Chromatography (FPLC). The affinity utilized is the affinity of the His tags to nickel.
  • The initial step is to equilibriate the system by adding 50 mL of the clear, colorless binding buffer to the superloop using a syringe. Flowpath was at the generic position Load.
  • 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.
  • 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.
  • 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 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 same steps were done for sample 2.
  • 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.



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