ResearchPage+-+Sadhana

Sadhana's Research
Express protein Set up crystal trays (use 24 well plates, start with condition listed in the PDB article and vary a little bit up and down in concentration) ICM Virtual Screening (maybe Vina screen too? - get Christina's help) Inhibition Assays Determine IC50 of compound Then compare best ICM ligand to best GOLD ligand in the wetlab

Week 14:
 * Since there were three proteins I collected from the FPLC, I did an enzyme assay for each one of them, however there was no enzyme activity. The OD reading was 0 for the most part
 * I also did an enzyme assay for Dr.B's PSTP sample prior to FPLC. Again, my enzyme was inactive.
 * I did an enzyme assay of all three increasing the concentration of enzyme I added to 10ng/ul. Since the solution didn't turn yellow, my enzyme was inactive.
 * Finally, I did an enzyme assay for my PSTP sample that was snap frozen, which didn't work as well.
 * [[image:Snapfrozen_sample-1.jpg width="350" height="219" caption="Figure 1: Concentration of PSTP that was snap frozen in -80 degrees Celsius"]]
 * [[image:Dr.B's_PSTP_before_FPLC-1.jpg width="356" height="223" caption="Figure 2: Concentration of Dr. B's PSTP before FPLC"]]
 * [[image:protein1-glycerol-1.jpg width="362" height="227" caption="Figure 3: Concentration of protein 1 in 20% glycerol"]]
 * [[image:protein3-glycerol-1.jpg width="366" height="229" caption="Concentration of protein 3 in 20% Glycerol - Possibly PSTP?"]]

Week 13:
 * Protein Expression
 * skipped characterization
 * FPLC
 * concentrated protein to 0.5ml and stored in 20% glycerol
 * [[image:elution1.PNG width="330" height="207" caption="Figure 1: Concentration of Elution 1 sample after purification"]]
 * [[image:Elution_2.PNG width="335" height="208" caption="Figure 2: Concentration of Elution 2 Sample after Purification"]]
 * [[image:FPLC.PNG width="800" height="240" caption="Figure 3: FPLC graph "]]

Week 12:
 * Enzyme Assay (2 Trials)
 * 1st trial - PTP1b as a control; used 60ul and 120ul of enzyme to test activity
 * PTP1b enzyme served as the positive control - turned yellow upon addition of pNPP substrate so enzyme works
 * PSTP did not turn yellow and the absorbance readings were 0.
 * 2nd trial - supplemented enzyme with manganese ions (final concentration is 1mM)
 * Tube A with 60ul of enzyme: 0.148 (absorbance)
 * Tube B with 120ul of enzyme: 0.107 (absorbance)
 * Need to do protein expression again...

Week 11: S - since the activity is below the control, the enzyme may not be active. I think the big bar in the second graph is an outlier. - May need to think about regrowing enzyme. Dr. B. (also may be worth comparing to PTP1b in same assay)
 * ACS
 * symBIOsis
 * Virtual Screening
 * Enzyme Inhibition Assay with and without TCEP

Week 10:
 * Virtual Screening-working on primary and secondary runs for MW-set_3D.sdf Chembridge Library
 * [[image:SB_excel.PNG width="491" height="507" caption="Figure 1: Best ranking list from first run of cb_306. "]]

Week 9:
 * Enzyme Test
 * [[image:SB_pstp.png width="685" height="580" caption="Figure 1: Absorbance of protein measured at 410nm"]]
 * [[image:SB_excelPSTP.JPG caption="Figure 2: Enzyme Activity - Absorbance at 410 nm vs Concentration of protein (nM). "]]
 * Concentrated protein after FPLC (below are the concentrations after FPLC)
 * [[image:afterFPLC-beforeconcentration-102411.jpg width="386" height="242" caption="Figure 2: Concentration of PSTP after it was diluted in buffer during FPLC - the concentration after Beer's Law calculation was 3.2uM."]]
 * [[image:afterFPLC-beforeconcentration-proteininquestion102411.jpg width="393" height="246" caption="Figure 3: Concentration of unknown protein detected by FPLC"]]
 * [[image:afterFPLC-afterconcentration2-102411-1.jpg width="399" height="250" caption="Figure 4: Concentration of PSTP after concentrating it to 16uM (After FPLC) - the concentration after Beer's Law calculation was 10.5 uM"]]
 * stored half of sample (.5ml) in 20% glycerol and snap froze the other half using liquid nitrogen
 * Crystallography workshop

Week 8:
 * Virtual Screened HF9, cd_306, MW-set libraries.
 * concentrated protein - combined elution 1 and 2 samples
 * [[image:concentratedElutionSB.jpg width="346" height="217" caption="Figure 1: After concentrating protein-before washing the "walls" of the filter. "]]
 * [[image:concentratedElution2SB.jpg width="350" height="219" caption="Figure 2: After concentrating protein - after washing the "walls" of the filter."]]
 * FPLC
 * [[file:20111021_SadhanaPSTP_FPLC.bmp]] Sadhana - embed this image instead of putting it as a link..... Dr. B

Week 7: - Need images or Data - Dr. B
 * Virtual screening on PSTP - screened the HF9 library
 * worked on pymol image of PSTP
 * Dried characterization gel
 * lab report

Week 6: (YEAH, Sadhana..... - good expression - Dr. B)
 * protein purification - combined soluble fraction of tube A and tube B
 * protein characterization (ran gel)
 * lane 1: protein ladder
 * lane 2: sample 1 (post induction) flask A
 * lane 3: sample 1 (post induction) flask B
 * lane 4: sample 2 (soluble fraction) tube A
 * lane 5: sample 2 (soluble fraction) tube B
 * lane 6: sample 3 (flow through)
 * lane 7: sample 4 (wash)
 * lane 8: sample 5 (Elution 1)
 * lane 9: sample 6 (Elution 2)

Concentrations of Elution Samples:





Week 5: - Sadhana - show some data - what were the pellet weights??
 * Made page gel
 * protein expression continued: lysis step 4

Week 4: (Sadhana - show a screen shot of your BLAST image instead of posting a link to a WORD doc. - Thanks,DrB.)
 * DNA sequencing results: Samples 2 and 6 matched completely. Sample 5 had a 99% match in both the forward and reverse sequence. I will be using Sample 6 for protein expression because it had the highest concentration. (37.8ng/ul)
 * Transformation (Bl21 competent cells) - How many colonies? - Dr. B (46 colonies on plate A and 221 colonies on plate B)
 * Protein Expression - What was the pellet weight? - Dr. B (3.08g and 2.96g)
 * [[image:vdsstream/SAM_0528.JPG width="299" height="248" caption="Figure 1: The top 2 plates are test plates with competent cells that had been left outside. The bottom two plates are my transformation plates with BL21 competent cells and my protein, PSTP. Plate A in the bottome ledt has 46 colonies and plate B on the bottom right has 221 colonies."]]
 * [[image:vdsstream/SAM_0532.JPG width="302" height="249" caption="Figure 2: My transformation plates and overnight culture. "]]

Week 3:
 * Centrifuged samples and retrieved the pellet (stored in -20 degrees Celsius)
 * Miniprep
 * Sent 3 samples (2,5,6) to DNA sequencing- The other samples had very low concentrations because I eluted with 100 ul of water as opposed to 50 ul of water.
 * Pymol Refresher
 * Virtual Screening Refresher (Completed Run 1)
 * [[file:concentration after miniprep.docx]]

Sadhana - looks good. - Dr. B

Week 2:
 * Preparation of pNIC-Bsa4 as Accepting Vector
 * Gel checked pNIC (not cut), so borrowed Adam's.
 * Cohesive End Generation and Transformation
 * Grew up culture and made master plate
 * [[image:vdsstream/sb_pnic.JPG width="363" height="273" caption="Figure 1: Lane 1-skip, Lane 2-uncut pNIC, Lane 3-cut pNIC...Bsa1 did not cut pNIC....possibly due to an error in PCR cleanup"]]

Week 1:
 * Ran secondary PCR on gel
 * Ran PCR Squared
 * PCR Cleanup on PCR Squared Sample
 * [[image:vdsstream/SB_PCR2.jpg width="491" height="314" caption="Figure 1: Concentration of secondary PCR prior to PCR cleanup"]]
 * [[image:vdsstream/sadhana_2nd_pcr.JPG width="332" height="254" caption="Figure 2: Secondary PCR prior to PCR cleanup; Lane 1: 100bp DNA ladder; Lane 2: Secondary PCR"]]
 * [[image:vdsstream/SB_PCRsquared1.jpg width="475" height="311" caption="Figure 3: Concentration of PCR squared sample after PCR clean-up."]]