Sandbox 323: Difference between revisions

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</StructureSection>

== Materials ==

- Buffers: Sodium Phosphate buffer, Cell Lysis Buffer Tris-HCl, 10X SDS-PAGE Buffer, Re-Suspension Buffer, 1X Wash Buffer, 1X Elution Buffer

- Solutions for SDS-Page: Coomassie Blue Stain, and Destain

- Pre-cast SDS-Page Gel

== Experimental and Results ==

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Fractions E1 through E6 were chosen to be run on an SDS-PAGE gel because they contained the highest amounts of protein. After running for an hour on 100-150 mV, the gel was stained and destained. The bands appeared around 30 kDa and fractions E2-E6 were pure.

Fractions E1 through E6 were chosen to be run on an SDS-PAGE gel because they contained the highest amounts of protein. After running for an hour on 100-150 mV, the gel was stained and destained. The bands appeared around 30 kDa and fractions E2-E6 were pure. The controls include running samples from before and after the column and wash.

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== Conclusions ==

The structure of 3DS8 was honed in on after matching the sequence with various other proteins in databases such as BLAST, SPRITE, and DALI. The sequence matches gave information about the active site and possible functions of the protein. The structure was then determined by the sequence in programs such as Chimera, InterPro, and SwissDock. The combination of this research suggested that the 3DS8 protein is a hydrolase. Gel electrophoresis confirmed that the protein was obtained as a dark band was visualized around 30 kDa. Various ligands that are common for hydrolysis were examined and modeled, and the best were used in the lab to gather data. Unfortunately, many of the ligands were on backorder and only PNPA was able to be used in activity assays. No data about the activity was obtained, as all the purified protein crashed out of solution before the assay could be run.

The structure of 3DS8 was honed in on after matching the sequence with various other proteins in databases such as BLAST, SPRITE, and DALI. The sequence matches gave information about the active site and possible functions of the protein. The structure was then determined by the sequence in programs such as Chimera, InterPro, and SwissDock. The combination of this research suggested that the 3DS8 protein is a hydrolase. Gel electrophoresis confirmed that the protein was obtained as a dark band was visualized around 30 kDa. Various ligands that are common for hydrolysis were examined and modeled, and the best were planned to be used in the lab to gather data. Unfortunately, many of the ligands were on backorder and only PNPA was able to be used in enzymatic activity assays. No data about the activity was obtained, as all the purified protein crashed out of solution before the assay could be run.

== References ==

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@@ Line 19: / Line 19: @@
 </StructureSection>
+== Materials ==
+- Buffers: Sodium Phosphate buffer, Cell Lysis Buffer Tris-HCl, 10X SDS-PAGE Buffer, Re-Suspension Buffer, 1X Wash Buffer, 1X Elution Buffer
+- Solutions for SDS-Page: Coomassie Blue Stain, and Destain
+- Pre-cast SDS-Page Gel
 == Experimental and Results ==
@@ Line 61: / Line 69: @@
-Fractions E1 through E6 were chosen to be run on an SDS-PAGE gel because they contained the highest amounts of protein. After running for an hour on 100-150 mV, the gel was stained and destained. The bands appeared around 30 kDa and fractions E2-E6 were pure.
+Fractions E1 through E6 were chosen to be run on an SDS-PAGE gel because they contained the highest amounts of protein. After running for an hour on 100-150 mV, the gel was stained and destained. The bands appeared around 30 kDa and fractions E2-E6 were pure. The controls include running samples from before and after the column and wash.
@@ Line 72: / Line 80: @@
 == Conclusions ==
-The structure of 3DS8 was honed in on after matching the sequence with various other proteins in databases such as BLAST, SPRITE, and DALI. The sequence matches gave information about the active site and possible functions of the protein. The structure was then determined by the sequence in programs such as Chimera, InterPro, and SwissDock. The combination of this research suggested that the 3DS8 protein is a hydrolase. Gel electrophoresis confirmed that the protein was obtained as a dark band was visualized around 30 kDa. Various ligands that are common for hydrolysis were examined and modeled, and the best were used in the lab to gather data. Unfortunately, many of the ligands were on backorder and only PNPA was able to be used in activity assays. No data about the activity was obtained, as all the purified protein crashed out of solution before the assay could be run.
+The structure of 3DS8 was honed in on after matching the sequence with various other proteins in databases such as BLAST, SPRITE, and DALI. The sequence matches gave information about the active site and possible functions of the protein. The structure was then determined by the sequence in programs such as Chimera, InterPro, and SwissDock. The combination of this research suggested that the 3DS8 protein is a hydrolase. Gel electrophoresis confirmed that the protein was obtained as a dark band was visualized around 30 kDa. Various ligands that are common for hydrolysis were examined and modeled, and the best were planned to be used in the lab to gather data. Unfortunately, many of the ligands were on backorder and only PNPA was able to be used in enzymatic activity assays. No data about the activity was obtained, as all the purified protein crashed out of solution before the assay could be run.
 == References ==