Ni 2 + Agarose Chromatography Lab Report

As mentioned previously, the two identified bands in the eighth and ninth lane, where EMG 9 is cultured, have high intensity and distinction. This can be explicated by the mutated _lacI_ gene found within this strain of _E.coli_. When this gene mutates, synthesis of _lac_ repressor will not transpire and activation of _lac_ operon will take place. Consequently, the non-mutated _lacZ_ gene within the EMG 9 strain will be transcribed and translated. Beta-galactosidase will become abundant resulting to increased intensity of bands. Therefore, it can be stated that there is direct relationship between protein abundance and intensity of the bands. Protein abundance will result to high band intensity (Hill, 1996).

coli bacteria was transformed with a gene that codes for green fluorescent protein. When constructed, the bacteria grew in the -pGLO LB agar plate, however, was unable to grow in the other plates. In the -pGLO LB/amp agar plate, there was neither glow or growth, which was expected, however, the other agar plates were all expected to show growth. Figure 1. picture from phone with blacklight Figure 2. picture taken with phone without

The goals of this lab report are to induce E.coli bacteria for producing protein of interest and determined the rate growth and protein concentration after inoculate the E.coli culture. In addition, protein visualizing and analyzing the success of IPTG induction, protein solubility and affinity chromatography demonstrated using SDS-PAGE. This report will also highlight the methodology of affinity chromatography that used to purify fused protein with poly His tag. A comparison between the advantage and disadvantage of using mammalian gene expression and bacterial expression system. Western bloating used to demonstrate the Mw of the protein interest and to analyze the immunoprecipitation of the protein of interest; including a brief

The plasmid first had to lose S65T-GFP so it could take up yEGFP. This was done by adding Pac1 and Asc1 to the plasmid to cut out the gene and leave the proper sticky ends for yEGFP to ligate to. A gel electrophoresis was then used to separate pWDH444 and S65T-GFP. The pWDH444 was cut from the gel electrophoresis and removed from the gel. After successful removal of the plasmid from the gel pWDH444 and yEGFP were ligated to create a new plasmid with the ideal GFP. The new plasmid, pWDH444-yEGFP was the ideal plasmid for yeast due to the enhanced GFP with a codon bias for yeast and it had double the light output of S65T-GFP. The difference in fluorescence was detected using a extra sensitive fluorimeter than normal and 495-nm glass, sharp cut-off filter. The filter reduced the amount of light shattering leading to an improved sensitive reading from the

The pEC86 plasmid with cytochrome c maturation system was co-expressed in E. coli cells to support the production of recombinant RpNiR (Han et al., 2012). Subsequently, the cells were harvested and yielded dark brown pellets, which were resuspended in buffer. The extracted mixture was loaded onto DEAE column and then a gel filtration column for purification. This has resulted in a light brown/red protein, which was expected to be rather dark red due to the presence of haem. The SEC uses UV-visible at specific wavelengths to detect the presence of RpNiR protein as shown in figure

In the past scientist have used the bacteria Escherichia coli (E. coli) in Synthetic Biology to allow the bacterial species to recognise and respond to light frequencies providing greater knowledge of complex biological systems (Levskaya et al. 2005). E. coli is a suitable chassis to express Green Fluorescent Protein (GFP) as the bacteria’s plasmid helps to establish GFP (Chavshin et al. 2013). The bacterial plasmid is a maltose-binding protein that helps E. coli and GFP fuse together and allows for detection of gene expression and controlling internal environments (Feilmeier et al. 2000). As GFP is a useful biological indicator due to its fluorescent structure (Mazzola et al. 2005) and through the ability of E. coli responding to light, GFP is easily expressed through the process of light stimulation (Lee, J et al. 2013). Through the unique structure and functions found within E. coli, it allows for successful expression of GFP making E. coli a suitable base organism.

Total proteins were prepared from cells of embryonic cortex at different ages (E10–18) and adult brain (2 months) with 500 µl of cell lysis buffer. Cell lysis buffer contains 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, 2 µg/ml aprotinin, and 1 mM PMSF. After homogenization on ice for 10 min, the supernatant of each sample was collected and stored in aliquots at -80 °C. For each sample, the protein concentration was determined by Bradford assay (Thermo Fisher Scientific). Cell lysate (20–50 µg) was resolved on 12% SDS-polyacrylamide gel electrophoresis and transferred onto PVDF or nitrocellulose membranes. The protein blots were blocked with 5% milk in TBS (10 mM Tris–HCl, pH 8.0, 150 mM NaCl) overnight at 4 °C and incubated with primary antibodies at a dilution of 1 to 1000–2000 in 5% milk prepared in TBS. The secondary antibody was donkey anti-mouse IgG-700 or anti-rabbit IgG-800 (LI-COR). Quantitative western blot was detected by the Odyseey CLx Infrared Imaging System according to the company’s instructions

30uL of the LB Buffer added to 30uL of each saved samples). Similar addition of LB Buffer (10uL) was made with the Protein Standards Ladder (10uL). After the addition of the LB Buffer to the appropriate samples, it was placed on a 95oC hot plate for 8 minutes (note: it was instructed to be for 5 minute). This was followed by the preparation of the gel setup (i.e. removing the tape at the bottom of the precast gel and adding the gels at the back and front of the apparatus gel). To this setup, 50uL of 10X SDS running buffer + 450uL of deionized water (making it a 1X Buffer) to the outer chamber was added. In the inner chamber, 150uL of 1X SDS (1X was made from the 10X SDS buffer, the same way by diluting it with a 1:9 ratio of deionized water) running buffer was added. With such setup and the 5 samples that was heated in 95oC, the lanes—starting with lane 1—were pipetted in the following order: 10uL of Protein Standards sample (lane 1), 20uL of “+IPTG with Elution Buffer” (lane 2 and 3), 20uL of “-IPTG with Elution Buffer” (lane 4 and 5), 20uL of “+IPTG Supernatant” (lane 6 and 7), and 20uL of “-IPTG Supernatant” (lane 8 and 9). After loading the gel in such manner, the start of running the gel was done at 200V (making note of it being stable initially), and it was stopped till the bromophenol blue bands reached ~1cm

Current antigen/antibody detection tests are based on agglutination reactions. Agglutination is the clumping of antigen-antibody complexes to form insoluble and visible aggregates. Agglutination occurs in two steps. The first step is sensitization, where the antibody present in the serum binds to the antigen sites on the RBC. The second step is “bridge formation”. Antibodies will form cross linking attachments to each other to bind the cells together. The strength and development of the attachments is affected by the class of antibody. IgM class antibodies are large pentameters, they can easily bridge the space between cells and with five arms the can form multiple attachments. Unlike IgG class antibodies, which are monomers and much smaller in size making it more difficult for them to bridge the gaps between cells

The induction process for the activation of rGFP was vital to this experiment, to ultimately determine the presence and molecular weight of rGFP from crude extract. T7 RNA Polymerase activates the T7 Promoter, by binding to it upstream of the rGFP gene, in the presence of IPTG, allowing the expression of rGFP. The expression results from the transcription of the rGFP gene that produces rGFP mRNA, which is then translated to produce the rGFP. By inducing the IPTG activation of the DNA regulatory sequence T7 Promoter, as seen in Figure 1, the presence of rGFP became apparent under the UV lights after the Ni+2 Agarose Affinity Chromatography washes and elutions. The G3 sample, three hours post-induction of the IPTG sample, had the highest fluorescence at 14901 RFUs.

However the rate of positive expression of p95HER2 reported in most of the studies used western blot technique was nearly 30% [14, 19, 26]. Also, immunofluorescense and immunoprecipitation techniques are impractical to be utilized for routine specimens, in addition, it highly expensive techniques [27]. To overcome this limitation Scaltriti et al. [5] designed immunofluorescence in FFPE tissues method and recorded 100% and 94% positive and negative predictive values respectively.

Used a fresh pipette tip for each sample and dispensed 10.0 µL of DNA ladder (M), 10.0 µL of R-, and 10.0 µL of R+ into designated wells.

The synthetic scFv(herceptin)-PE-stxa chimeric gene was cloned in a pET28a expression vector. Recombinant vector pET28a-scFv(herceptin)-stxa was digested by BamHI and xhoI restriction enzymes and 1683-pb DNA sequence was obtained. Then recombinant vector pET28a was transformed into E. coli strain BL21(DE3) by elcteroporation. The transformed colonies were cultured in 5 mL of Lysogeny broth (LB) containing 20 mg/mL kanamicyn at 37 °C. When OD600 reached 0.6–0.8, 1 mM IPTG was added. The culture was incubated at 37 °C for 6 h. Then, the cells were collected by centrifugation at 3,000×g/10 min. Then, expression of scFv(herceptin)-stxa) chimeric construct was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). Different IPTG concentrations (0.5, 0.6, 0.8, and 1 mM), and various induction duration (2, 5, 7, 16 H), at two separate culture temperature (30 and 37 °C), were tested for optimization of protein expression.

It is of strong interest to molecular biologists because of the strong light emission of 508 nm under UV light. UV wavelengths excite the fluorophore, contained in the Ser - Tyr - Gly sequence near GFP's N-terminus, and a bright green fluorescence is given off as a result (Niwa et al., 1996). GFP can be successfully tagged to many proteins without disturbing their function (Feilmeier et al., 2000). This allows scientists to exploit the protein for its usefulness in marking proteins in vivo. GFP tagging has many applications. It can be used to study gene expression by inserting gfp after a promotor in the plasmid, so that if expressed, the colony expressing the gene of interest can be identified by a green fluorescence of GFP under UV light (Feilmeier et al., 2000). Specific cellular organelles can be tagged, such as the endoplasmic reticulum, to visualize protein localization and networking inside the living cell. GFP can also be used to monitor cofactor levels within a cell in real time. For example, when GFP was tagged to proteins that contain a receptor sensitive to Ca2+, they interacted in a way so that the fluorescence of the fusion protein was proportional to the calcium ion concentration with less than a 1 second lag in the cell (Romoser et al., 1997). Throughout the decades, GFP mutants have been created to give brighter emission and different wavelengths. Researchers can track separate cell pathways at the same time, since different proteins can

To analyze the folding of recombinant TGF-β1, purified protein was electrophoresed on native gel, under non-reducing condition and stained with the silver stain. Electrophoresis of the purified TGF-β1, under non-reducing condition, confirmed the presence of dimeric form of the TGF-β1, as a 25 kDa band on the non-denaturing gel (Figure 5 ). Here, there was no trace of a monomeric TGF-β1 band (13.5 kDa). This result indicated that the mature form of TGF-β1 is probably folded properly, and secreted as a dimer into the culture medium.