Proteins from Mammalian Cells Extraction

Proteins from Mammalian Cells Extraction Introduction: The aim of this practical was to generate protein material from Caco-2 cells and measuring the amount collected by using a bicinchinonic assay (BCA assay). The Caco-2 cell line is widely used in laboratories since it has very similar morphologic characteristics to normal enterocytes. This provides the cells with similar functions such as transport of nutrients and enterocytic differentiation when cultured in a monolayer in vitro. Therefore, Caco-2 cells have been shown to be a suitable model system to investigate the structure and function of the small intestinal epithelium (Ismael J. HIDALGO). By breaking up these cells, the collected components can be analysed in further experiments. This is usually done by using a detergent based solution to lyse the cell but other methods can also be used such as electrical lysis. Once this process has undergone, a homogenous extract of the broken-down cell is obtained and various techniques can be performed to analyse the components. The first use of the broken-down cell material in this practical was in a BCA assay to help measure the amount of protein yielded from the extraction step. The BCA technique is very commonly used in laboratories to measure the total protein concentration of a sample by comparing it to a protein standard. This method is popular because it can not only accurately determine the protein concentration of most sample types, but can also have various other applications such as measuring column fractions after performing an affinity chromatography or studying protein to protein interactions. The method measures the concentration of protein by analysing the reduction of Cu2+ to Cu1+ in an alkaline environment (known as the biuret reaction) combined with the colorimetric detection of Cu1+ by the bicinchoninic acid. This produces a signal that can be read by a spectrophotometer. The results can then be analysed to determine the concentration of the protein sample (Measure of protein using bca, PK SMITH RI KROHN). Protein extraction Materials Pipettes and tips Deionized water NaCl Trizma base lot#SLBH1724V (sigma) opened on 13/12/2013 Triton-x-100 lot#48H0208 (sigma) Sodium dodecyl sulphate Protease inhibitor Caco-2 cell culture plate (2 wells) Cell scraper Centrifuge tube 3x 1,5mL, 1x 50mL Plate reader Method Firstly, a buffer was prepared by adding 0.174g of NaCl with 0.303g of Tris to a 50mL plastic tube. 2,5mL of triton-x-100 and 0,5mL of Sodium dodecyl sulphate were then added to the tube. Water was finally added to create a final volume of 25mL. The buffer pH was then measured using a standard pH meter. Hydrogen ions were slowly added combined with a continuous monitoring of the pH change to obtain a final pH of 8,6. Buffer calculations: NaCL (Mr=58) 120mM needed => (58g/M)x(0.12M) =6.96g 6.96g/40=0.174g for 25mL Tris (Mr=121) 100mM needed => (121g/M)x(0.1M) =12.1g 12.1g/40=0.3025g for 25mL Triton-x-100 1% needed from 10% stock solution => 25mL/100 (to get to 1%) =0.25mL 0.2510 (10% stock solution) =2.5mL Sodium dodecyl sulphate 0.2% needed from 10% stock solution => 25mL/500 (to get to 0.2%) =0.05mL 0.05mlx10 (10%stock solution) =0.5mL Next, 1mL of protease inhibitor solution was prepared by adding 10 µl of protease inhibitor to 1mL of the buffer prepared earlier in a 1.5mL Eppendorf tube. The tube was then placed on ice. The next step was the addition of 200 µl of protease inhibitor buffer each to 2 wells of the caco-2 cells followed by vigorous scraping at the bottom of the well using a cell scraper. The cell suspension was then removed and placed into a microfuge tube and placed on ice. The tube containing the cell suspension was left to incubate on ice for 30 minutes receiving a resuspension by inverting the tube every 10 minutes. The tube was then centrifuged at 13000 rpm for 5 minutes.   After noticing that the sample needed more centrifugation, the tube was centrifuged for another 3 minutes at 13000rpm. The supernatant was then collected into a fresh tube. BCA assay Materials Pipettes 1x 96 well plate Bovine serum albumin (BSA) 2mg/mL Deionized water BCA reagents A and B Ependorf tubes Extracted protein Method Firstly, 6 standards were prepared by diluting Bovine Serum Albumin (BSA) (2mg/mL) with water as followed: Table 1. Preparation of standards volumes Concentration (mg/mL) BSA (mL) Water (mL) 0 0 100 0.25 12.5 85.5 0.5 25 75 1 50 50 1.5 75 25 2 100 0 25  µl of each standard was loaded on the 96 well plate in duplicate as shown below. 5 µl of cell extract mixed with 20 µl of water to create a 1/5 dilution of the extracted protein was then loaded in 3 separate wells. Also, 2.5 µl of cell extract mixed with 22.5 µl of water to create a 1/10 dilution of the extracted protein was also added to three separate wells of the plate. Table 2. 96 well plate distribution 1 2 3 4 5 6 7 8 9 10 11 12 A 0 0 Sample 1 (1/5) Sample 1 (1/5) Sample 1 (1/5) B 0.25 0.25 Sample 2 (1/10) Sample 2 (1/10) Sample 2 (1/10) C 0.5 0.5 D 1 1 E 1.5 1.5 F 2 2 G H The next step was to prepare a working reagent which was made by mixing 5mL of BCA reagent A with 100 µl of BCA reagent B. 200 µl of this BCA reagent was added to all the wells containing standards and samples. And the plate was then incubated at room temperature for 30 minutes. Finally, the plate was read at 540nm on a plate reader. The remaining cell extract was stored at -20C for further experiments. Results After cell extraction and the BSA assay, the sample absorbance results from the plate were as followed: Standards: Table 3. Standards absorbance Standards (mg/ml) 0.000 0.250 0.500 1.000 1.500 2.000 Absorbance Date: 23/01/2017 0.077 0.338 0.495 0.828 1.083 1.438 Date: 23/01/2017 0.083 0.348 0.500 0.799 1.056 1.469 Mean 0.080 0.343 0.498 0.814 1.070 1.454 Standard deviation (n=2) 0.004 0.007 0.004 0.021 0.019 0.022 CV % (n=2) 5.303 2.062 0.711 2.521 1.785 1.508 Table 4. Standards mean absorbance recapitulative Standards Concentration Mean Abs 1 0 0.08 2 0.25 0.343 3 0.5 0.498 4 1 0.814 5 1.5 1.07 6 2 1.454 Using these results, a standard curve was plotted (Figure2.). Figure 1. Standard Curve (Absorbance over concentration) Samples: Table 5. Samples absorbance results and mean Absorbance Results Sample 1 (1/5) (n=3) Sample 2 (1/10) (n=3) 0.529 0.319 0.539 0.332 0.536 0.368 Mean 0.535 0.340 By extrapolating the known absorbances obtained from the samples on the standard curve, a final absorption can be calculated. Note that the dilution factor is considered to create an end concentration and the mean of both samples was calculated to finalise the measurement of extracted protein (Table 6.). Table 6. Final extracted protein concentration Sample 1(1/5) sample 2 (1/10) End concentration (mg/ml) absorbance 0.535 0.34 Sample 1 sample 2 mg/ml Concentration (mg/mL) 0.609 0.312 3.04 3.12 Mean 3.08 Total error (n=2) 0.057 CV % (n=2) 1.837 Finally, after the extraction of the protein from the caco-2 cells and the BCA assay we can affirm that the amount of protein yielded had a concentration of 3.08 mg/ml. Discussion This practical shows the essential mechanisms involved in breaking down a cell to analyse its material. This is firstly done by lysing the cell to release its contents. The most common method in doing so is by using a detergent-based solution such as sodium dodecyl sulphate (SDS). Sodium dodecyl sulphate is used in many methods such as in gel electrophoresis (SDS-PAGE see practical 2) or nucleic acid extraction. The structure of SDS gives it an amphiphilic property, meaning it is both hydrophilic and lipophilic, both essential properties to be used as a detergent. It works by disrupting non-covalent bonds of proteins which produces dissociation of protein complexes. This results in the solubilisation of cell membrane proteins for example. There are different types of detergents, some can be denaturing reagents such as SDS, and others can be non-denaturing. The other detergent used in this practical is Triton X-100 which is a non-denaturing, non-ionic detergent. This detergent contrib utes to maintaining the protein structures to a minimum (size and charge) (thermos fischer SDS). Another important step in the extraction of cell material is the centrifugation of the cell suspension following cell lysis. The centrifugation step is used to separate the components of a homogenate, in this case, the cell suspension. The extract is rotated at high speeds, creating a separation of the components by size and density. The larger the component, the greater centrifugal force will be applied, hence they will move the most rapidly. By altering the speed of centrifugation, different components can be isolated. Using this technique, we can collect the components by forming a pellet. If the pellet is impure, as it was during the experiment, repeated centrifugation may improve its quality (fractioning of cells, molecular biology of the cell 4th edition). After successfully obtaining the cell extract and performing the BCA assay, a standard curve can be plotted. But how accurate and precise is this curve? This depends on the quality of the results from the BCA assay standards. In other words, the precision of these. The precision of the standards was determined by testing two replicates on the plate. And was expressed as a coefficient of variation percentage (CV% where CV=standard deviation (SD)/mean) (Desvignes). Figure 2. Comparison of the coefficient of variations of the standard duplicates As shown above, the CV% for all standards were very low (usually acceptable below 20%). This means that the precision of the results was good and that the standard curve is precise(Desilva)(EMEA). In this experiment, the cell line that was used were Caco-2 cells derived from a tumour of the human gut epithelium and are a model of enterocytes. (The human intestinal epithelial cell line Caco-2; pharmacological and pharmacokinetic applications) A monolayer of caco-2 cells exhibit very similar characteristics to the cells found in the small intestine due to its morphology. For example, they express microvillus hydrolases and nutrient transporters commonly found in the small-intestine. This makes them very useful in mimicking the gut in a laboratory setting. Conclusion         Ã‚   By using a detergent based solution, it is possible to break up cells to collect their material for further analysis. The quantification of the material can be achieved by performing a BCA assay which involves various techniques such as centrifugation followed by plotting a standard curve using standards prepared. This material can then be used in further experiments to analyse their components. In this experiment, the Caco-2 cell line was used, this cell line was derived from a tumour of the human gut epithelium and share various similarities with the cells commonly found in the small intestine.