ResearchPage+-+Shutian

Week Fourteen
I did protein purification and characterization on Monday. Figure 1: Absorbance of elution 1. The concentration is 45.

Figure 2: Absorbance of elution 2. The concentration is 10.80. This is much lower than the absorbance of elution 1 because most of the proteins was already washed into the elution 1 tube. Figure 3: The protein band is at about 45 kDa. The protein PP2Ctg is supposed to be from 42 - 52 kDa. Even though the expression is relatively weak, the correct protein was expressed successfully. Lane 1 – PageRuler Ladder Lane 2 – Cell lysate before induction Lane 3 – Cell lysate after induction Lane 4 – soluble fraction Lane 5 – Flow through (waste) Lane 6 – Wash Lane Lane 7 – Elution 1 Lane 8 – Elution 2 Figure 4: Top 10 ligands from two of the libraries. These fitness scores are pretty low. At the same time, none of these satisfied the Lipinski's Rule of Five.

Week Thirteen
I did my second run for the ChemBridge library.

Week Twelve
My virtual screening for ChemBridge- diversity3D.sdf is still running. I have been working on the virtual screening.

Week Eleven
I have been checking on my virtual screening, and the ChemBridge- diversity3D.sdf is still running. I have finished protein expression. My pellet weight was 2.81.

Week Ten
This week, I did my PCR clean-up and saved the samples in -20 degree Celsius. I am also doing my protein expression. My virtual run for ChemBridge-diversity3D.sdf is still running.

Week Nine
I have been working on the Virtual Screening. I have done the 2nd run for the cb_306_3d.sdf and the HF9PlatesPlates5_9.sdf. However, the ChemBridge-diversity3D.sdf is still on its first run because it is such a large library.

Week Eight
I also ran the primary runs for the HF9 and the Chembridge libraries.

Primer Overlap Primer Design
 * tgpp 1 || ATGGATGTACCTCCGACCATCCACGTACCGCTCCC || 35 ||
 * tgpp 2 || GTCGGTGAAGATCGCCGCGTCGAACGCTGGGTAAGAGGTTGGCGGGAGCGGTACGTGGAT || 60 ||
 * tgpp 3 || CGGCGATCTTCACCGACATCGGTGGCCGTAAACACCAGGAGGATCGTTTCACCCTGTGCC || 60 ||
 * tgpp 4 || ACCGAAGAACGCGCAGTCGTCACGACCCGGAACCAGCTGTGGGCACAGGGTGAAACGATC || 60 ||
 * tgpp 5 || ACTGCGCGTTCTTCGGTGTTTTTGATGGTACCGTTGGTGATTTCGCGTCTGAAAACGTTA || 60 ||
 * tgpp 6 || ACGCCGGAGAAGAGATGAGCTGCGGTACAACCAGATCTTTAACGTTTTCAGACGCGAAAT || 60 ||
 * tgpp 7 || TCATCTCTTCTCCGGCGTGGCAGGAAGTTACCGAAATGCTGCGTTCTGATGTTCCGGCAA || 60 ||
 * tgpp 8 || CGCCTGGTCGAGGAGTTGCGGCAGTTTTTCGTCTACTTCAGTTGCCGGAACATCAGAACG || 60 ||
 * tgpp 9 || AACTCCTCGACCAGGCGGTTGACGATATGTACAAAAACGCAGACAACGAACTGGTTAAAA || 60 ||
 * tgpp 10 || GGTAGAAGACGCGTAGTCTTTGTTCAGCTGTTCGCACATTTTAACCAGTTCGTTGTCTGC || 60 ||
 * tgpp 11 || AAAGACTACGCGTCTTCTACCTCTGTGACCGCTGTTCTGGCGAAAGGCTTCGTTGCGGTT || 60 ||
 * tgpp 12 || GGTCTCAACACCCATGGCGATGCGAGAGTCACCGAGGTGACCAACCGCAACGAAGCCTTT || 60 ||
 * tgpp 13 || GCCATGGGTGTTGAGACCCCGAATGGCCTGAACTGCGAGTTCCTCACCGTTGACCACAAA || 60 ||
 * tgpp 14 || CCGTTACGCATGATGCGGAGCTTTTCGTGCGGCATGTCCGGTTTGTGGTCAACGGTGAGG || 60 ||
 * tgpp 15 || CCGCATCATGCGTAACGGTGGTTCTGTTGAATACCTGCACAATCATAACAACAAACCTTT || 60 ||
 * tgpp 16 || CGAGATTTACGGAAAGAGAAGTCACCACCACGGATGAAAGGTTTGTTGTTATGATTGTGC || 60 ||
 * tgpp 17 || GACTTCTCTTTCCGTAAATCTCGTGGTGAACAGCCAATGCAACTGCAATACTCTCGTGCG || 60 ||
 * tgpp 18 || TGGTTAGAGAGACCATACATTTTGAGGTCTTTACCACCGAACGCACGAGAGTATTGCAGT || 60 ||
 * tgpp 19 || CAAAATGTATGGTCTCTCTAACCAGCCGGACGTTCGCGTTGTTCGTGTTACCCCGCAACA || 60 ||
 * tgpp 20 || ATGACGTCCCAGAGACCGTCGGTCGCGAGGATCATTACACGATGTTGCGGGGTAACACGA || 60 ||
 * tgpp 21 || CGGTCTCTGGGACGTCATGTCTGCTGCGCAAGCAGTCGAAATTGCTATGCAAGCGCGTCA || 60 ||
 * tgpp 22 || CAGGGTCATTTCTACCAGCGCCTGGGCTGGGTTACGACCTTCCTGACGCGCTTGCATAGC || 60 ||
 * tgpp 23 || CGCTGGTAGAAATGACCCTGGCGGAACAGCAGTCTCGCAATCAGTCCGCTGACAACACGG || 60 ||
 * tgpp 24 || GTCGGTTTTCTTGAAAAAGACGGTCATGGCCGTGTTGTCAGCGGAC || 46 ||

Week Seven
I ran the primary and secondary PCR and they came out very well. I have also finished the Primer Design for the overlap. Lane 1: Empty Lane 2: 100 bp ladder Lane 3: Primary PCR Lane 4: Secondary PCR Lane 5: Run over from the secondary PCR

Week Six
I have been working on the PCR primer overlap this week. I have stored the primary and secondary PCR in the -20 __degrees__ refrigerator. The PCR squared still needs to be run.

Week Five
The Virtual Screening Refresher was done running. Through the analysis by using PyMOL, the top 10 ligands were observed and compared to MTX.

(1) Output 5, Ligand 1 (2) Output 1, Ligand 1 (3) Output 2, Ligand 1

(4) Output 1, Ligand 2

(5) Output 2, Ligand 2

(6) Output 2, Ligand 3

(7) Output 4, Ligand 1

(8) Output 3, Ligand 1

(9) Output 3, Ligand 2

(10) Output 5, Ligand 2

MTX



2D Images for the top 3 bind ligands

1) 2) 3)

Week Four
I was working on the Virtual Screening Refresher. The second run took a while because I was waiting for the blades.

Week Four
2nd PCR: I accidentally added DNA ladder into Sample A/lane 1. But the result was still able to show up for the sample.

I also finished the Pymol Refresher. This is the 2H2Q molecule with the two chains specifically labeled. 3CLQ RMS = 1.126 (111 to 111 atoms) 1U72 3HBB

Week Three
The results of the Submit DNA sequencing: This is blasted against the human genome. This is blasted against the protein.

RE Digest with the bands clearly shown. Lane1: Empty Lane 2: 1 kb ladder Lanes 3 & 4: Uncut plasmid Lane 5: PvuII Lane 6: EcoRI Lane 7: EcoRI and PvuII

PCR try #3 Lane 1: Empty Lane 2: 100 bp Ladder Lane 3: Sample A Lane 4: Sample B Lane 5: Sample C Lane 6: Sample D

Week Two
Gel observed on September 9, 2011

Lane 1: EcoRI digest reaction (This was repeated due to the collapse of the wall) Lane 2: 1 kb ladder Lane 3: EcoRI digest reaction Lane 4: PvuII digest reaction Lane 5: EcoRI and PvuII digest reaction Lane 6: Uncut plasmid (pGBR22 426.3 ng/ul)

Lane 7: 100 bp ladder Lane 8: Sample A Lane 9: Sample B Lane 10: Sample C Lane 11: Sample D Lane 12: 1 kb ladder (This was repeated due the collapse of the wall)

This gel electrophoresis failed because the gel was not solidified enough. As a result, the walls of the wells collasped and the samples smeared together.

Week One
Human sequencing Nucleotide sequencing Reading frame 3 is the right reading frame. (ORF Finder) Comparison of the Purple Protein to pGBR22 Plasmid Gel Outlook

This week, I also submitted the DNA sequence with the plasmid pGBR22 and the primers T7 and T7term. First try of PCR failed, possibly due to contamination. (Gel observed on September 7, 2011)

Lane 1: 100 bp Ladder Lane 2: Sample A Lane 3: Sample B Lane 4: Sample C Lane 5: Sample D Lane 6: Empty