Team:Slovenia/METHODS and PARTS/notebook/protein

8/23/2010 – 8/29/2010

Production of protein: cCFP_link_HivC (5 hours, 37°C, 160 RPM). Protein accumulated mainly in inclusion bodies  Cell lysates Protein analyzed by SDS-PAGE and western blot using antibodies agains His-Tag. Further protein purification of  protein in solubilized inclusion bodies was attempted by Ni-NTA column, and elution by increasing concentration of imidazole (25mM, 50mM, 100mM, 250mM, 500mM). After concentration using Centricon with the cutoff, the protein was re-analyzed by SDS page and Western blot.

8/30/2010 – 9/5/2010

Production of proteins: Pbs2, Gli1, Zif268, Tyr, Jazz, Blues (5 hours, 37°C, 160 RPM). After production, proteins accumulated in inclusion bodies or in the supernatant was analyzed by SDS-PAGE.

9/6/2010 – 9/12/2010

Production of  HivC protein was repeated, this time in 0,5 L follwowing the protocol 12 h incubation at 25 °C, 160 RPM. More protein was expected in supernatant (that would make purification and SPR measurements a lot easier). Production of the protein was repeated the next day, at 37°C. Lysis buffer with NaCl and Zinc-sulphate was made. After lysis we tried to get rid of DNA with help of streptomycin but unfortunately that was unsuccessful. NaCl was added to the buffers for protein purification. Inclusion bodies after purification were concentrated and tested with SDS-PAGE and Western blot.

9/13/2010 – 9/19/2010

Spectra of purified proteins were measured and HivC, Jazz and Zif268 were dialyzed. After dialysis,  spectra of the samples were measured again and concentration of proteins was determined. In addition, the folding of the proteins was examined by circular dichroism (CD) Samples were than taken to Biotehnical faculty for SPR analysis. SPR showed binding of Zif268 protein. Production of the rest of the proteins (PBS, Blues, Gli) was finished and SDS-PAGE and western blot was made. After that samples were dialyzed and concentarted.

9/20/2010 – 9/26/2010

Concentration of proteins was determined by A280. After that  proteins were analyzed by SDS-PAGE.  Concentration of proteins was also determined by bicinchoninic acid (BCA) assay. Production of Gli, Zif268 and Jazz was repeated.

9/27/2010 – 10/3/2010

All zinc-finger proteins were dialyzed agains new dyalisis buffer. Proteins were analyzed by A280 and Bradford assaybecause BCA reagent reacted with dialysis buffer. Samples were also tested by SPR.

10/4/2010 – 10/10/2010

Even though results on SPR were very good, protein production was repeated. This time we ree-attempted purification on a Ni-NTA column to obtain protein with the higher purtity. Bradford. SDS-page and western blot showed that purification of the proteins  was unsuccessful.  gli1 and hivC proteins were purifes as before.

10/11/2010 – 10/18/2010

After purification samples were dialyzed and concentrated. Protein concentration was determined by Bradford assay. SDS-PAGE gel proved that HivC protein was purified and and was sent for testing on SPR. Producion of all six zinc fingers, Nictal and three new zinc fingers with longer binding sites was performed. After SDS-PAGE analysis, there was no production of new zinc fingers and nictal. Purification of Gli, HivC, PBS, Zif 268 was repeated. All the proteins were purified on Nicei-NTA colon. Low yield of proteins production, as detemined by Bradford assay was achieved. In addition, no proteins were detected on SDS-PAGE after dyalisis.

10/19/2010 – 10/26/2010

No Zif and Jazz proteins were detected by SDS-PAGE and western blot. Dialysis was repeated, however, majority of the protein precipitede out of solution. I tried new protocol for fast refolding of the proteins with no success.