Protein Concentration Determination Report PDF

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Lexie Orr Cell Biology Lab Professor Crum February 10, 2020 Determination of Concentration of Unknown Protein #16 Introduction: The goal of this lab was to determine the unknown concentration of protein in our sample, which was number sixteen, and another goal was create a standard curve of BSA to compare our protein sample to. This was done by a series of dilutions and Bradford assays to obtain a dilution factor by comparing it the absorbance levels of BSA. Bradford assays are used for protein concentration determination of the unknown lysosome along with direct absorbance. As well, this experiment follows closely along with Beer’s law and helps to allow us to further elaborate on it. Methods: For the first part of this lab my partners and I had to create a BSA standard dilution to determine if we needed to dilute our sample and by how much. The first part was done by taking 40uL of 1.0mg/mL BSA in well A8 then continuously pipet down 20uL into each well until A2 after that to get to a dilution of 0.0625 mg/ml. We then performed the same technique with our unknown sample in wells B2 through B8. After we added 200 ul of Bradford reagent to wells A1 through A8 and B1 through B8 and waited five minutes to let the dye change colors. Then we had to find which color blue from wells A1 to A8 corresponded with the middle of the curve and then tried to find that same color blue in wells B1 to B8 and whichever one it matched to, told us what we had to dilute our sample to. We then performed a serial and discrete dilution of BSA from 0.0625 mg/ml to 1.00 mg/ml and then ran an assay in triplicate using the Bradford Assay. This was done by preparing five dilutions of protein standard with PBS solution and aliquoting 10ul of diluent and then 10ul of unknown. Results: The goal of this experiment was to find the protein concentration in sample number 16, which we tested by serially diluting from 0.0625 mg/ml to 1.0 mg/ml and assayed in triplicate using the Bradford assay, as well as running a discrete assay. From comparing our unknown to a dilution of the BSA standard we hypothesized that our unknown sample was diluted to 1/16 so we diluted it to that factor for the serial dilution. However, when we ran the discrete assay with a 1/16 dilution our results were not good so we ran it again with a ¼ dilution. For our discrete assay we obtained an average absorbance reading (A595) of 0.120 nm for unknow #16. For the serial assay we got a negative average absorbance reading (A595) of -0.025nm which was not in range. Once our data was collected we created a graph for both assays comparing average BSA absorbance readings at (A595) vs BSA concentration from 0.0625 to 1.0 mg/mL to have a standard curve and equation to base our concentrations on for our unknown. After that we calculated where our

sample would fall based on absorbance readings. For the discrete dilution we calculated that our concentration would be 0.93 mg/ml and for the serial assay we got 0.43 mg/ml. BSA Absorbance Absorbance Concentratio (A595 nm) 1 (A595 nm) 2 n (mg/mL) 0.0625 0.054 0.039 0.125 0.231 0.104 0.25 0.350 0.133 0.5 0.535 0.521 1 0.693 0.734 Unknown 0.142 0.102 Diluted 1/16 Figure 1: Discrete Assay Data Table

Absorbance (A595 nm) 3

Average

Standard Deviation

0.065 0.112 0.350 0.517 0.633 0.117

0.053 0.149 0.278 0.524 0.687 0.120

0.0131 0.0711 0.1253 0.0095 0.0508 0.0202

Discrete Assay 0.8 0.7

f(x) = 0.66 x + 0.08 R² = 0.92

Absrobance

0.6 0.5

BSA (0.625-1.0 mg/mL) Linear (BSA (0.6251.0 mg/mL))

0.4 0.3 0.2 0.1 0

0

0.2

0.4

0.6

0.8

1

1.2

BSA Concentration mg/mL

Figure 2: Absorbance vs BSA concentration standard curve for Discrete Assay BSA Absorbance Absorbance Concentratio (A595 nm) 1 (A595 nm) 2 n (mg/mL) 0.0625 0.040 0.067 0.125 0.127 0.185 0.25 0.274 0.293 0.5 0.713 0.622 1 0.832 0.907 Unknown -0.049 -0.008 Diluted 1/4 Figure 3: Serial Assay Data Table

Absorbance (A595 nm) 3 0.083 0.203 0.298 0.616 0.940 -0.02

Average

0.063 0.172 0.288 0.650 0.893 -0.025

Standard Deviation 0.022 0.040 0.013 0.054 0.055 0.021

Absorbance (595nm)

Serial Assay 1 0.9 0.8

f(x) = 0.89 x + 0.07 R² = 0.94

0.7 0.6 BSA (0.625-1.0 mg/mL) Linear (BSA (0.625-1.0 mg/mL))

0.5 0.4 0.3 0.2 0.1 0

0

0.2

0.4

0.6

0.8

1

1.2

BSA Concentration (mg/mL_

Figure 4: Absorbance vs BSA concentration standard curve for Serial Assay Discussion: The purpose of this lab was determine the concentration of our unknown protein sample and we determined it to be 0.93 mg/ml from our discrete assay but got a value of 0.43mg/ml for our serial assay which are two very different values. Our serial assay graph had less deviations when it came to looking at the standard error bars for standard deviation but I think we ran into some problems when running our serial assay because we got negative numbers for our unknown absorbance so it was not in range with our BSA standard absorbance readings. What I believe happened is that our dilutions of our sample were incorrect. This could have been due to us pipetting incorrectly or even doing the dilution of our unknown incorrectly. We knew protein was present in our reactions because there was a color change when the reagent was added. Furthermore, this experiment follows Beer’s law which states that the quantity of light absorbed by a substance is directly proportional to the concentration of the substance which applies to this protocol because we are comparing absorbance of BSA to its concentration graphically and got a directly proportional line. The reason we used a standard curve is to one obviously represent a relationship between two quantities but to also determine a value of an unknown quantity from one that is more easily measure. If the points on our standard curve did not make a straight line that would just mean we did our dilutions incorrectly. Also, if our unknown concentration was higher than the highest point on the standard curve that would mean we would have to dilute it more. It would be okay to use values from a standard curve that was prepared earlier in the day for a protein determination 3 hours later as long as all other factors were the same. Lastly, we showed standard deviations for our data in order to show our far our values were from mean value of the data set and to also show how spread our data was. Overall my partners and I prefer the discrete assay because we obtained better results....