Quantitative Determination of CD14 IN RAW264-7 Macrophage Cells - Research Paper Example

Quantitative determination of CD14 IN RAW264.7 Macrophage cells NAME STUDENT NUMBER 1685 word Quantitative determination of CD14 IN RAW264.7 Macrophage cells Abstract The determination of the quantity of a protein is essential in the estimation of its activity. This experiment uses the western blotting, spectrophotometric and TNFA ELISA to quantify the CD14 proteins that are present in the sample. The western blotting process is performed first to confirm the presence of CD 14 cells in the sample of raw macrophages. The expression levels of these cells were then analyzed after exposure to both Nitrite Oxide and peroxide. CD14 plays an important role in the detection of bacterial LPS. It is also key in host defense through macrophages which express it. This experiment involves quantification of CD14 in RAW264.7 macrophages. Hypothesis statement: This experiment indicates that Raw 264.7 macrophages express CD 14 (LPS PRR). These experimental techniques are considered most useful in any molecular laboratory in the world. The experimental protocol begins with western blotting followed by spectrophotometric assay and final TNFA ELISA. The sample used in western blotting differs from the other processes in that whole cell lysate was used during western blotting whereas the other assays use the supernatant. Every experiment done was done against a control. Standard curve construction was used in the data presentation and analysis. This presentation is aided by a computer software which automatically analyses and outputs the data. The research finding indicates the presence of NO and peroxides. Moreover, the SDS-PAGE stains blue for Coomassie die. These together with TNFA confirms the presence of CD14. This makes it right to conclude that CD14 proteins are present in macrophages and are responsible for some of the receptor mediated reactions. Introduction This experiment is concerned with quantification of Nitric Oxide and peroxide that is found in tissues. Usually, Western Blot analysis is considered one of the most important techniques in the molecular laboratory. This technique is used as an analytical approach for the detection of proteins. In simple terms, western blotting involves the transfer of biological samples from gels to a known membrane. Subsequently, these samples are then detected on the surface of the membranes. The technique of western blotting is sometimes called immunoblotting due to the fact that it often involves the use of antibodies which help to detect their own antigens (Van Stappen et al., 2015). The specificity of the antigen-antibody interaction is of great importance in the identification of the wanted proteins. The amount of Nitric Oxide and Peroxides are important in the identification of tissues. It is a fact that Nitric Oxide, for example, would occur in high quantities tissues that are responsible for neurotransmission, host defense, and even vascular homeostasis. This means that Nitric Oxide is suitable for determination of proteins especially given the fact that it absorbs spectrophotometric light. On the other hand, Hydrogen peroxide also occurs in tissues and it can be used to demonstrate the catabolic activity of tissues. Moreover, TNFA ELISA would also prove useful for the quantification of biological samples. The quantity is measured by determining the amount of the amount of target that is bound between two pairs of antibody (Janova et al., 2016). Therefore in a nut shell, the aim of this experiment is to determine the quantity of protein in the samples used. CD14 expression in the macrophages would be an important marker in this experiment. The experimental design entails the quantitative determination of CD14 levels, Nitric Oxide and Peroxides. Important is to note the fact that expression level of proteins varies significantly in biological samples. This calls for the need to do multiple tests in this experiments. The western blotting process would first involve the confirmation of the presence of CD 14 cells in the sample of raw macrophages. The expression levels of these cells would then be analyzed after exposure to both Nitrite Oxide and peroxide. CD14 plays an important role in the detection of bacterial LPS. It is also key in host defense through macrophages which express it. This experiment involves quantification of CD14 in RAW264.7 macrophages. The hypothesis: This experiment indicates that Raw 264.7 macrophages express CD 14 (LPS PRR). MATERIAL AND METHODS Materials and reagents P20 single channel pipette SDS-PAGE equipment Turbo Blotter 5X Sample buffer 12 5 15 well acrylamide mini gel Prestained protein molecular weight standards SDS-PAGE running buffer Wypall X60 white sheets soaked in Tris/CAPS anode & cathode transfer Blocking buffer Wash buffer PBS Colour development reagent Reagent reservoirs Multichannel pipette P200 single channel pipette 96-well flat-bottomed enzymatic assay plate 2 % sulphanilamide in a 5% phosphoric acid 0.2% naphthyl ethylenediamine dihydrochloride (NEDD) 0.1% Sodium Nitrite (Nitrite Standard) Complete media Reagent A: 25 mM ammonium ferrous (II) sulfate, 2.5 M H2SO4 Reagent B: 100mM sorbitol, 125 µM xylenol orange in water. 1 mM hydrogen peroxide Capture antibody solution Procedure I. CD14 WESTERN BLOT Sample was prepared or the SDS-PAGE SDS PAGE was loaded and run SDS-PAGE was transferred using Trans-Blot Turbo system Membranes were blocked Blocking Incubation of membranes using primary antibody Incubation with secondary antibody Blot Development. II. Quantitative Nitric Oxide Spectroscopic Assay Cell culture supernatant was prepared were incubated with buffer poly IC, LPS or Poly IC and LPS for 15 minutes and then snap frozen at -80 degrees. Nitrite standard reference curve was prepared for quantification of NO in the sample. Griess reaction was used to measure the amount of nitrite. After the reaction absorbance was measured at 30 minutes and values used to plot standard curve after calculation of results with the aid of computer based curve fitting software. III. Quantitative Peroxide Spectroscopic Assay. Cell culture supernatants were prepared by incubating with E. coli in different conditions then snap frozen. Standard curve was generated using peroxide serial dilution Assay procedure A single channel P20 pipette was used to add 20 µl of sample or standard to the wells in a microplate. 200 µl of WR was added to each well using a multichannel pipette. This was pipetted 10 times up and down for thorough mixing. The assay reaction was incubated for 20 minutes at room temperature. After 1 hour, the absorbance was measured at 595 using plate reader. The concentration of peroxide was then calculated in the sample by referencing the assay absorbance to the standard curve. IV. TNFA ELISA Prior to anything, the ELISA plates were coated with TNFA Antibody and reagents put at room temperature. TNFA ELISA was performed using standard procedure provided in the manual and results calculated. Results SDS-PAGE shows that all sample lanes are equally loaded. Purple markers were at 25 and 75. There were no protease inhibitors – lower molecular weight of CD14 – due to it being polyclonal epitope – rather than one protein. Some epitopes would be digested, but some would stay around – this was due to proteolysis. Results of the SDS-PAGE was shown in figure 1 below. Figure 1. SDS-PAGE Photographic results. Figure 1. This is a photograph of the SDS-PAGE from the Western Blot analysis of the sample Figure 2. Graphical representation of NO results The results show that the Optical Density of the sample increases with Concentrations. The r value for this experiment is 0.9995. The absorbance readings are an indication of the presence of NO. The absorbance which is a direct indication of NO is seen to increase with an increase in concentration. The presence of NO in the sample confirms the activity of CD14 proteins in these cell samples. Figure 3. Graphical representation of quantification of peroxides The optical density of the Peroxides increases as the concentration of the sample increases. Figure 4. Graphical representation of TFNA. TNFA graphical representation of the Absorbance and concentration indicates an increase until an optimal level is reached. This indicates the activity of the TTFNA increases until it reaches an optimal level where it remains constant. From statistical analysis, the r value for this data is 0.9975. The rate of activity varies slightly from the different samples done in the laboratory. Discussion CD14 proteins are greatly responsible for the upregulation of LPS. The western blotting assay involves the determination of the binding affinity of these two proteins. The western blot assay was conducted using the whole cell lysate of RAW264.7 whereas the Nitrite Oxide, Peroxide and TNFA ELISA analysis were done using supernatant of the sample. The same bands in the SDS-PAGE was an indication of the presence of CD14 in the whole lysate. This was a confirmation of the quantity of CD14 in both the samples provided. It is a fact that these levels would vary depending on the sample being monitored. The presence of the bands, therefore, confirms that the protein sample would respond to LPS. The rate of production of peroxides, Nitric Oxide and Tumour Necrosis Factor differ significantly. However, the increase in the absorbance of the samples with an increase in the concentration of the sample indicates the increase in activity of the CD14 to its binding site (Rossi et al., 2015). From the data, it is evident that none of the treatment groups altered the expression levels of CD14 in the biological sample used. It is, therefore, true according to the hypothesis that Raw 264.7 macrophages express CD 14 (LPS PRR). Research findings have always indicated that CD14 is critical for MyD-88 dependent signal transduction. The protein is useful in the adaptive response of humans. The presentation of antigen, CD 14 is critical to not only the quality of the response but also the type of this response by the immune system. The innate immune system is used for this determination process. During inflammation usually, the innate immune system produces macrophages as a response to the inflammatory process. The macrophages in normal situations would increase the rate of pathogen phagocytosis when active by DAMPs. Moreover, the secretions rates of cytotoxic agents such as Peroxides and cytokines such as Tumour Necrosis Factor α. The experiment herein investigated the innate activation of macrophages from RAW264.6 cells by the use of PAMPs and DAMPs in the absence of immunoglobulin. The presence of NO, Peroxide, and TNFA in the results confirms the innate activation of the macrophages in the sample. Both western blot, spectrophotometric and ELISA assay confirms the activation process. The pathways of CD14 in this experimented are activated by the LPS, Poly IC. Further experimental studies should consider studying of the possible inflammatory pathways that are present in various pathogenic interactions (Van Stappen et al., 2016). References Janova, H., Böttcher, C., Holtman, I. R., Regen, T., van Rossum, D., Götz, A., ... & Gronke, K. (2016). CD14 is a key organizer of microglial responses to CNS infection and injury. Glia, 64(4), 635-649. Rossi, E., Basso, D., Zambon, C. F., Navaglia, F., Greco, E., Pelloso, M., ... & Bozzato, D. (2015). TNFA Haplotype genetic testing improves HLA in estimating the risk of celiac disease in children. PloS one, 10(4), e0123244. Van Stappen, T., Brouwers, E., Tops, S., Geukens, N., Vermeire, S., Declerck, P. J., & Gils, A. (2015). Generation of a highly specific monoclonal anti-infliximab antibody for harmonization of TNF-coated infliximab assays. Therapeutic drug monitoring, 37(4), 479-485. Read More