Proteins from Mammalian Cells Extraction

Proteins from Mammalian Cells bloodIntroductionThe aim of this practical was to generate protein bodily from Caco-2 electric cellular phones and measuring the add up collected by development a bicinchinonic curb (BCA substantiation).The Caco-2 cell cable is widely apply in laboratories since it has very similar morphologic characteristics to general 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 good example system to investigate the grammatical construction and function of the teensy intestinal epithelium (Ismael J. HIDALGO).By breaking up these cells, the collected components domiciliate be analysed in further experiments. This is usually done by using a detersive averaged dissolver to lyse the cell but other methods can too be employ such as electrical lysis. Once this demonstrate has undergo ne, 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 veridical in this practical was in a BCA assay to help assess the amount of protein yielded from the extraction step. The BCA technique is very commonly used in laboratories to measure the total protein assiduousness of a sample by comparing it to a protein pattern. This method is popular because it can not only accurately construe the protein denseness of just about sample types, but can similarly have various other applications such as measuring tugboat 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) have with the colorimetric detection of Cu1+ by the bicinchoninic acid. This produces a signal that can be read by a spectrophotometer. The results can consequently be analysed to determine the concentration of the protein sample (Measure of protein using bca, PK SMITH RI KROHN).Protein extractionMaterialsPipettes and tipsDeionized piddleNaClTrizma base lotSLBH1724V (sigma) opened on 13/12/2013Triton-x- deoxycytidine monophosphate lot48H0208 (sigma)sodium dodecyl sulphateProtease inhibitorCaco-2 cell culture plate (2 wells)Cell scraper spin tube 3x 1,5mL, 1x 50mLPlate readerMethodFirstly, a lover was wide-awake 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 atomic number 11 dodecyl sulphate were then added to the tube. pee was last(a)ly added to create a final volume of 25mL. The buffer pH was then measured using a ensample pH meter. enthalpy ions were s unhopefully added combined with a continuous monitoring of the pH form to obtain a final pH of 8,6.Buffer calculationsNaCL (Mr=58) 120mM requisite = (58g/M)x(0.12M) =6.96g 6.9 6g/40=0.174g for 25mLTris (Mr=121) 100mM needed = (121g/M)x(0.1M) =12.1g 12.1g/40=0.3025g for 25mLTriton-x-100 1% needed from 10% stock solution = 25mL/100 (to get to 1%) =0.25mL 0.2510 (10% stock solution) =2.5mLSodium dodecyl sulphate 0.2% needed from 10% stock solution = 25mL/500 (to get to 0.2%) =0.05mL 0.05mlx10 (10%stock solution) =0.5mLNext, 1mL of proteinase inhibitor solution was prepared by adding 10l of proteolytic enzyme inhibitor to 1mL of the buffer prepared earlier in a 1.5mL Epp set asideorf tube. The tube was then displace on ice.The next step was the addition of 200l of protease inhibitor buffer each to 2 wells of the caco-2 cells followed by vigorous slit at the bottom of the well using a cell scraper. The cell suspension was then removed and laid into a microfuge tube and placed on ice.The tube containing the cell suspension was left to incubate on ice for 30 proceedings receiving a resuspension by inverting the tube every(prenominal) 10 minutes. The tube w as then centrifuged at 13000 rpm for 5 minutes. after(prenominal) 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 assayMaterialsPipettes1x 96 well plateBovine serum albumin (BSA) 2mg/mLDeionized waterBCA reagents A and BEpendorf tubesExtracted proteinMethodFirstly, 6 standards were prepared by diluting Bovine Serum Albumin (BSA) (2mg/mL) with water as followed put over 1. planning of standards volumestightness (mg/mL)BSA (mL)Water (mL)001000.2512.585.50.52575150501.575252100025 l of each standard was loaded on the 96 well plate in duplicate as shown below. 5l of cell extract mixed with 20l of water to create a 1/5 dilution of the extracted protein was then loaded in 3 set forth wells. Also, 2.5l of cell extract mixed with 22.5l of water to create a 1/10 dilution of the extracted protein was also added to trey separate wells of the plate.Table 2. 96 well plate dispersal123456789101112A00Sample 1(1/5)Sample 1(1/5)Sample 1(1/5)B0.250.25Sample 2 (1/10)Sample 2 (1/10)Sample 2 (1/10)C0.50.5D11E1.51.5F22GHThe next step was to prepare a working reagent which was made by mixing 5mL of BCA reagent A with 100l of BCA reagent B.200l 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.Resultsafter cell extraction and the BSA assay, the sample absorbance results from the plate were as followedStandardsTable 3. Standards absorbanceStandards (mg/ml)0.0000.2500.5001.0001.5002.000Absorbance while 23/01/2017 0.0770.3380.4950.8281.0831.438Date 23/01/20170.0830.3480.5000.7991.0561.469Mean0.0800.3430.4980.8141.0701.454Standard deviation (n=2)0.0040.0070.0040.0210.0190.022CV % (n=2)5.3032.0620.7112.5211.7851.508Table 4. Standards mean absor bance recapitulativeStandardsConcentrationMean Abs100.0820.250.34330.50.498410.81451.51.07621.454victimization these results, a standard rick was plotted ( realize2.).Figure 1. Standard bender (Absorbance over concentration) SamplesTable 5. Samples absorbance results and meanAbsorbanceResultsSample 1 (1/5) (n=3)Sample 2 (1/10) (n=3)0.5290.3190.5390.3320.5360.368Mean0.5350.340By 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 concentrationSample 1(1/5)sample 2 (1/10)End concentration (mg/ml)absorbance0.5350.34Sample 1sample 2mg/mlConcentration (mg/mL)0.6090.3123.043.12Mean3.08Total error (n=2)0.057CV % (n=2)1.837Finally, after the extraction of the protein from the caco-2 cells and the BCA assay we ca n affirm that the amount of protein yielded had a concentration of 3.08 mg/ml. handlingThis practical shows the essential mechanisms involved in breaking down a cell to analyse its material. This is firstly done by lysing the cell to let go 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 practica l is Triton X-100 which is a non-denaturing, non-ionic detergent. This detergent contributes to maintaining the protein structures to a negligible (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 postgraduate speeds, creating a separation of the components by size and density. The larger the component, the greater motor(a) force will be applied, hence they will move the most rapidly. By altering the speed of centrifugation, different components can be isolated. utilise this technique, we can collect the components by forming a pellet. If the pellet is impure, as it was during the experiment, iterate centrifugation may improve its quality (fractioning of cells, molecular biology of the cell quaternate edition).After successfully o btaining 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 duplicatesAs 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 neoplasm of the piece 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 reveal very similar characteristics to the cells found in the small intestine out-of-pocket to its morphology. For example, they express microvillus hydrolases and nutrient transporters commonly found in the small-intestine. This makes them very effectual in mimicking the gut in a laboratory setting.Conclusion 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.