Difference between revisions of "Team:UCLA/Notebook/Recombinant Expression/15 July 2015"

Latest revision as of 07:06, 17 July 2015

Plan for Growing Up a New Liter of Tamura

Evaluation of What We've Done So Far

The results of the BCA assay and SDS PAGE indicate that while the extracted Tamura proteins are pure, the yields are quite low. When fractions 3-5 from the previous IMAC batch purification procedure were pooled together and the contents run through a Pierce concentrator, only a little remained in the filter, showing that the protein was suspended in a high volume of buffers and solvents, rendering it very dilute. The concentrated fraction had a surprisingly low weight per volume ratio, estimated to be 0.12 w/v %. Therefore, to obtain greater quantities of concentrated protein of interest, and to better assist the members on the Materials Processing Team with their co-spinning objectives, we will grow up a new liter of Tamura. This time, the correct amount of Lysis Buffer will be added to re-suspend the cell pellets. This will make a significant difference in preventing our Tamura proteins from becoming solvated and diluted in excess solutions. We will also save reagents as quantities of lysozyme, deoxycholic acid, and DNAse required will all be reduced.

Also, when using the Histidine beads last time, there was concern that not enough Tamura protein was adhering to the beads due to an overwhelming presence of excess contaminant proteins binding to the beads instead. When purifying the Tamura proteins this time, the proteins will be suspended in a lot less lysis buffer, resulting in a concentrated solution of Tamura even before using a Pierce concentrator. The increased concentration of Tamura in the volume of solvent will increase the chances of Tamura binding to the beads, limited only by the binding capacity of the beads themselves. This will increase the effectiveness of our purification protocol.

@@ Line 287: / Line 287: @@
 </html>
 <u>Plan for Growing Up a New Liter of Tamura</u>
 '''Evaluation of What We've Done So Far'''
-The results of the BCA assay and SDS PAGE indicate that while the extracted Tamura proteins are pure, the yields are quite low. When fractions 3-5 from the previous IMAC batch purification procedure were pooled together and the contents run through a Pierce concentrator, only a little remained in the filter, showing that the protein was suspended in a high volume of buffers and solvents, rendering it very dilute. The concentrated fraction had a surprisingly low weight per volume ratio, estimated to be 0.12 w/v %. Therefore, to obtain greater quantities of concentrated protein of interest, and to better assist the members on the Materials Processing Team with their co-spinning objectives, we will grow up a new liter of Tamura. This time, the correct amount of Lysis Buffer will be added to re-suspend the cell pellets. This will make a significant difference in preventing our Tamura proteins from becoming solvated and diluted in excess solutions. We will also save reagents as quantities of lysozyme, deoxycholic acid, and DNAse required will be reduced. Also, when using the Histidine beads last time, there was concern that not enough Tamura protein was adhering to the beads due to large presence of excess contaminant proteins binding to the beads instead. When extracting the Tamura proteins this time, the proteins will be suspended in a lot less lysis buffer, resulting in a concentrated solution of Tamura even before using a Pierce concentrator. The increased concentration of Tamura in the volume of solvent will increase the chances of maximum Tamura binding to the beads, limited only by the binding capacity of the beads themselves.
+The results of the BCA assay and SDS PAGE indicate that while the extracted Tamura proteins are pure, the yields are quite low. When fractions 3-5 from the previous IMAC batch purification procedure were pooled together and the contents run through a Pierce concentrator, only a little remained in the filter, showing that the protein was suspended in a high volume of buffers and solvents, rendering it very dilute. The concentrated fraction had a surprisingly low weight per volume ratio, estimated to be 0.12 w/v %. Therefore, to obtain greater quantities of concentrated protein of interest, and to better assist the members on the Materials Processing Team with their co-spinning objectives, we will grow up a new liter of Tamura. This time, the correct amount of Lysis Buffer will be added to re-suspend the cell pellets. This will make a significant difference in preventing our Tamura proteins from becoming solvated and diluted in excess solutions. We will also save reagents as quantities of lysozyme, deoxycholic acid, and DNAse required will all be reduced.
+Also, when using the Histidine beads last time, there was concern that not enough Tamura protein was adhering to the beads due to an overwhelming presence of excess contaminant proteins binding to the beads instead. When purifying the Tamura proteins this time, the proteins will be suspended in a lot less lysis buffer, resulting in a concentrated solution of Tamura even before using a Pierce concentrator. The increased concentration of Tamura in the volume of solvent will increase the chances of Tamura binding to the beads, limited only by the binding capacity of the beads themselves. This will increase the effectiveness of our purification protocol.