The Protein Interactions Between Gst, Gst Ebna1 And 6xhis Usp7 Through Affinity Chromatography

Affinity chromatography involves the property of bio-recognition for the separation of proteins based on the reversible interaction between the protein and the specific ligand bound to a chromatography matrix. It enables us to purify bio-molecule on the basis of its biological function and its chemical structure. The goal of the experiment was to gain hands-on experience in protein purification by using AKTA FPLC system. The IgG antibody contained in the mouse antiserum was purified by protein G affinity chromatography using a HiTrap protein G HP column. The chromatogram was then obtained and analyzed to ensure the separation of IgG antibody. Such purification of the protein before sample preparation is usually done to avoid any undesired interactions.

3.6.1. BAP (Benzyl amino purine) stock solution (2mg/ml): 20mg of BAP being weighed and dissolved completely in 1N NaOH to a final total volume of 10 ml with autoclaved double distilled water to obtain a stock concentration of 2mg/ml was prepared and stored in clean autoclaved vials at -4°C.

In Figure 3, there is a collected data with the total washes and the elution fraction with the RFUs from the Ni+2 Agarose column. E3 had the highest elution fraction with 8554 RFUs. In the Bradford assay, E3 had the highest protein amount with 65.5ug. The specific activity for E3 was about 1.28E5 RFUs/mg. Overall, in the Figure 3, the E1-E6 had most of the rGFPs due to being bonded in Ni+2. W1-W6 rGFP is much smaller than the E1-E6 amounts.

Second, in order to further confirm the information about characteristics and function of the targeting protein that we have

Protein samples separated by size via SDS-Page can be identified using mass spectroscopy; which will require proteins from the gel to be treated using trypsin; which is an enzyme that digests proteins. Assuming that samples were treated and storaged properly, the digestion of peptides begins with the rehydration of proteins using trypsin, followed by the incubation of proteins using a

The second objective is to study the acceptor specificity, using different fragments of the large acceptor of ST6Gal-I.

For the second part of the experiment, one had to use the knowledge learn from viewing protein molecules in FirstGlance in Jmol to analyze the protein PDB ID: 4EEY. The analysis of this protein was done using the RSCB protein data bank (PDB) at (http://www.rcsb.org/pdb/home/home.do).2

The supernatant is the cytoplasmic fraction. The crude nuclei pellet was resuspended in Extraction Buffer (20 mM HEPES, pH 7.9, with 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, 25% (v/v) Glycerol and 1 mM DTT, 1 mM PMSF, 2 μg/ml leupeptin, 2 μg/mL pepstatin A), gently shaked for 30 minutes and centrifuged for 5 minutes at 20,000 × g.

Through a series of experiments we are trying to demonstrate that we are able to express and purify a recombinant GFP in E. coli strain BL21(DE3)<pRSETA-GFPUV using a Ni2+ agarose affinity chromatography method. The findings showed that one the highest amount of activity came from elution 3 with 15144 RFU’s. The estimated protein amount in Elution 3 was about 49ug using the Bradford assay, and specific activity of 309061 RFU/mg, the SDS page showed that E3s purity was about 33 %. The SDS Page showed faint bands at both, 28kDa and 32 kDa, and western Blot analysis, showed that there was definite rGFP found in the E3, as well as all the Fractions.

The de-ubiquitinase (DUB) will cleave this precursor into free monomer ubiquitin molecules and can be activated and attached to the target protein through ATP dependent process called ubiquitination with the help of activating enzyme E1, conjugating enzyme E2, and ligase E3. Through forming (iso)-peptide, thioester, or ester bond between carboxyl groups of C-terminal glycine 76 on ubiquitin and the ε-amino group of the lysine, cysteine, serine/threonine on the substrate, respectively, the poly-ubiquitin chain is formed [3]. Based on the different internal Lysine (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63) and on the proximal ubiquitin, the (iso)-peptide bond can be form with another ubiquitin, distal mono-ubiquitin [4, 5]. Consequently, seven conventional poly-ubiquitin chains resulting in different 3-dimentional shapes explaining its different characteristics inherited upon binding to the target protein [5, 6]. With ranging more than 500 putative E3 ligases exist in human, the E3 ligase will tag the poly-ubiquitin chain to the target protein with high specificity

Results: Enzyme digestion analysis, PCR and DNA sequencing results showed the target gene was inserted correctly into the vector. The expressed protein was purified successfully via Ni-NTA affinity chromatography and its molecular weight

MP is generally extracted in intermediate or high-ionic-strength buffer, which is required to disrupted the myofibrils enough to release myosin, therefore, known as “salt-soluble proteins”.

At this point the GFP is bound to Ni2+ in the matrix of the Ni-NTA column, however it is not alone. Many other proteins and cellular components may have bound to the column as well due

NetPhos 2.0 analysis projected 9/11/11 serine, 2/3/2 threonine and 5/7/7 tyrosine phosphorylation sites for GS01/GS02/GS03 proteins respectively. The homology modelling of the enzyme shows 12 identical subunits, arranged in two layers of 6. The secondary structure of GS consisted of 7 alpha helix and 15 beta strands. The binding residues, predicted by the RaptorX binding web server, and the corresponding ligands for the three different GS proteins are given in Table 3.

UPLC-qTOF-MS/MS Waters was used to determine the amino acid sequence of the peptide moiety. The apparatus conditions were: ESI (electrospray ionization), positive mode; capillary temperature, 300oC; capillary voltage, 40 V and carrier gas flow 500 L/h. The lipopeptide and fractions were loaded to qTOF-MS directly without hydrolysis. Tandem-MS was done by collision-induced dissociation-mass spectrometry (CID-MS) using argon as collision gas and collision energy of 40 eV.