Protein Assay: The Pierce BCA Protein Assay (Thermo Scientific) is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein concentration. A series of standard solution of Bovine Serum Albumin (BSA) ranging from 0-2000 µg/ml was prepared from a stock solution of 2 mg/ml BSA. 25ul of diluted crude (1:500, 1:250), desalted (1:100, 1:50), and 6 peak fractions from cibarcon blue column (1:10, 1:5) were loaded in microplate along with 175ul of BCA working reagent. Microplate was incubated for 30min at 370C and then the absorbance was measured at 562nm.
After the substrate solution was added, five drops of the enzyme were quickly placed in tubes 3, 4 and 5. There were no drops of enzyme added in tubes 1 and 2 and in tube 6 ten drops were added. Once the enzyme solution has been added the tubes were then left to incubate for ten minutes and after five drops of DNSA solution were added to tubes 1 to 6. The tubes were then placed in a hot block at 80-90oC for five minutes. They were then taken out after the five minute period and using a 5 ml pipette, 5 ml of distilled water were added to the 6 tubes and mixed by inversion. Once everything was complete the 6 tubes were then taken to the Milton Roy Company Spectronic 21 and the absorbance of each tube was tested.
b. Attached to the secondary antibody is an enzyme such as peroxidase or alkaline phosphatase. These enzymes can metabolize colorless substrates (sometimes called chromagens) into colored products. After an incubation
This technique separates Rubisco samples based on their size. The electrophoresis has a positive and a negative end. Positive charge proteins are loaded from the positive end and migrate towards the negative end. Negative charge proteins are loaded from the negative end and migrate towards the positive end (Sakthivel & Palani, 2016). The sample that contained the highest molecular weight of Rubisco will travel the shortest distance on the gel while the protein with the smallest molecular weight will travel the longest distance (Sakthivel & Palani, 2016). The size proportion of each Rubisco molecule correlates with the distance traveled. Rubisco will be in its purest form after running through SDS-page since each technique will increase the purity of the protein. If the salting out, the ion exchange and the SDS-page protein isolation techniques are performed on protein Rubisco, then it is purified and separated by solubility, charge, and size. The rationale of this experiment is to isolate the purest form of Rubisco so that it can perform carbon fixation at an optimal
Figure 1 contains gel electrophoresis for protein samples. The lanes were labeled from 1 to 10 from the right to the left. Lane 1 contained the ladder fragment. Lane 2 contained the filtrate. Lane 3 contained the S1 sample. Lane 4 contained the P1 sample. Lane 5 contained the P1 medium salt sample. Lane 6 contained the P1 high salt sample. Lane 7 contained the S2 sample. Lane 8 contained the P2 sample. Lane 9 contained the P2 medium salt sample. Lane 10 contained the P2 high salt sample.
The homogenates provided were made by homogenizing tissues in a sucrose phosphate buffer in a 1:20 ratio. The protein concentration in bovine cells was measured by diluting the homogenate with a 1:5 ratio; 50 microliters of homogenate and 200 microliters of water. Then 5 known protein concentration samples which were 0.4, 0.8, 1.2, 1.6, 2.0 mg/ml of bovine serum were used to determine absorbance with a spectrophotometer. Two additional samples were made; one was blank and the other was for the specific homogenate sample. Then 3 microliters of bradford assay reagent, which indicates the amount of protein present
3. The tubes are allowed to incubate in a 37˚C water bath for 1 hour. The final pH of the solutions is tested and the amount of protein digestion is estimated using a scale of (+++), (++), (+), and (-) by
Observe how protein biomarkers are assayed by ELISA assays beginning with serum samples, and learn how to perform these assays. (Mice sera)
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
Briefly, for SDS-polyacrylamide gel electrophoresis (SDS-PAGE), cell lysate (20 ug protein per lane) samples were loaded onto 10-12 lane Tris-Glycine 4-20% mini-gels (Invitrogen) (Zhang et al., 2009). Following electrophoresis, separated proteins were laterally transferred to polyvinylidene fluoride (PVDF) membranes (iBlot semi-dry transfer system) at a constant voltage of 12 V for 9 min at ambient temperature. After electrotransfer, the membranes were blocked for 1 hr at ambient temperature in 5% non-fat milk prepared in tris-buffered saline containing 0.05% Tween-20 (TBST) followed by incubation at 4o C for 3 hr or overnight with primary antibodies [mouse anti-αII-spectrin (Biomol) or rabbit anti-GFAP (Abcam) at 1:2000 dilution in TBST-5% non-fat milk). This was followed by three washes with TBST, a 2 hr incubation at ambient temperature with a secondary antibody (goat anti-rabbit or anti-mouse IgG conjugated to alkaline phosphatase (Novagen) and developed with substrate BCIP-NBT reagent (KPL). Housekeeping proteins, β-actin and/or carbonic
Colorimetric assay is a process of determining a concentration of a solution based on absorbance of light. The purpose of this lab is to determine if the Bradford assay is an accurate way to determine an unknown concentration of two samples of protein. The Bradford assay is done by measuring wavelength of light passing through a cuvette filled with Bradford dye and concentrations of PBS and proteins. After the cuvettes are mixed they are placed into a spectrophotometer to measure wavelength. The wavelength given will be used to plot a standard curve based on concentration (x-axis) and wavelength (y-axis). The standard curve is then used to measure an educated guess on the concentrations of unknown protein concentrations. We hypothesized that if we use the Bradford assay and colorimetric spectrophotometry we can determine an accurate concentration of two unknown concentrations of proteins. The results of this lab failed to reject our hypothesis based on accurate measurements of protein concentrations. The standard curves are drawn with a linear increasing slope. The Bradford assay is an accurate way to demine the concentration of an unknown concentration.
The labels associated with the resulting proteins from previous steps were rinsed and removed. USP7 was purified and utilized in sedimentation trials in varying concentrations. Absorbance analysis was carried out after the samples were centrifuged. EBNA1 was purified, ubiquitinated, and combined with USP7, which was purified in previous steps. SDS-PAGE was used to disrupt the protein interaction and separate them. The separated fragments were then used in a western blot, and analyzed after probing. The affinity of USP7 for EBNA1 and p53 was tested using a stepwise combination of titration, incubation, and fluorescence analysis. Lastly, amino acid residues of EBNA1and USP7 were studied by gel filtration and size-exclusion chromatography.
The last part of this procedure, antibodies will be used to detect one specific protein from others on the membrane. Incubate the membrane with 10 ml of primary antibody for 10-20 minutes and then place on a rocking platform. Rinse the membrane quickly after with wash buffer on a rocking platform. After three minutes is up, discard the wash and incubate the membrane with 10 ml of secondary antibody for 5-15 minutes on the platform. Rinse the membrane again shortly after and wash the membrane for three minutes. Next, discard the wash and add 10 mL of HRP color detection reagent. Incubation will occur after this for 10-30 minutes. Once it is done, rinse the membrane twice with distilled water and blot dry.
The topics of these experiments are both important processes known as identification and quantification. They were also used to review and introduce new lab methods such as titration and the Bradford assay. The identification was used to determine the identity of unknown amino acid #11 and the quantification was used to determine the concentration of unknown protein #11. The identity of amino acid #11 was found to be lysine, and the concentration of protein #11 was calculated to be 0.51 ± 0.019 ug/uL with a 3.67% error, reflecting accuracy in the results.
The intracellular or secreted proteins of interest are then isolated and purified as transformants are scaled-up.