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BSCI330 Laboratory Notebook

2 Table of Contents Lab 1: An Introduction to the Study of Cells (9.9.20) ….…….…………….……..…………Pages 1-5 Lab 2: Micropipette Technique Review and Spectrophotometric Methods (9.16.20) ………Pages 6-11 Lab 3: Protein A: Properties and Methods of Isolation (9.23.20) …………...…..…………..Pages 12-14 Lab 4: Protein B: Protein Quantification and Gel Electrophoresis (9.30.20) ...………….…..Pages 15-20 Lab 5: Enzyme Kinetics (10.7.20)……………………………………………………………Pages 21-27 Lab 6: Cell Communication: Receptors and Ligands (10.14.20)…………………………….Pages 28-33

1 Lab Exercise 1: An Introduction to the Study of Cells Date: 9.9.2020 Objective: The objective of this lab is to get familiarized with basic microscopy concepts such as resolution and magnification, as well as demonstrate why microscopy is important and useful when studying cells. Bright field microscopy, using an ocular micrometer, phase contrast microscopy, and the examination of living cells and tissues will be touched upon. Bright field and phase contrast microscopy are very versatile and widely used techniques in biological research. Bright field microscopy shines light onto a specimen point to which absorbs the light differently because the density and thickness of the specimen are space-variant—"making the energy of light after passing through the specimen,” space-variant1. This method of microscopy is the “number one choice whenever minimization of expenditure or implementation difficulties are main concerns.” 1 Additionally, phase contrast microscopy is extremely useful when researching transparent, non-lightabsorbing specimens because it produces a contrast to make them more visible. This technique “highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens (like tissue cells) and does not suffer from the phase halos typical of phase-contrast images.” 2 Works Cited: 1. Stanislav K. (2005). Light microscopy in biological research. Biophysical journal, 88(6), 3741. https://doi.org/10.1529/biophysj.105.060046 2. Centonze Frohlich V. (2008). Phase contrast and differential interference contrast (DIC) microscopy. Journal of visualized experiments : JoVE, (17), 844. https://doi.org/10.3791/844 Pre-lab: I-A: 1) As magnification power increases, the working distance will decrease. This is because in order to magnify the image of a sample, the lens will need to get closer to the sample; thus, decreasing the working distance. 2) The field of view diameter will decrease as magnification power increases. The field of view diameter decreases by a factor of about 2.5 from 4x to 10x, and about a factor of 10 from 4x to 40x.

2 2 I-B: Objective Lens

NA

R Values (nm)

R Values ( m)

4x

0.1

5500

5.5

10x

0.25

2200

2.2

40x

0.65

846

0.85

I-C: Cell Organelle

Average Size

Resolvable with a 40x?

Nucleus

7-10 m diameter

Yes

Plasma membrane

4-5 nm thick

No

Mitochondrion

1-3 m long, 1 m wide

Yes

Chloroplast

5 m long, 2 m wide

Yes

Microfilaments

5 nm thick

No

Lysosome

0.2-0.5 m diameter

No

3: At 100x magnification: 100 μm = 10 ocular units = 10 μm/ocular unit 4: At 400x magnification: 100 μm = 40 ocular units = 2.5 μm/ocular unit

Materials: 

Microscope



Stage micrometer slide



Fixed paramecium slide



Fixed human blood smear slide



Fixed amoeba slide

3 2 Procedures: 1. Used a microscope equipped with a stage micrometer slide to observe and analyze fixed slides of paramecium, human blood smear, and amoeba.

Procedure detailed in “A. Bright Field Microscopy” in the BSCI 330 Lab Manual. Cantemir-Stone, Carmen, editor. “An Introduction to the Study of Cells.” BSCI 330 Laboratory Manual: Cell Biology and Physiology - University of Maryland. Macmillan Higher Education, 2020, pp. 13-14.

Results: Table 1. Average size and standard deviation of various organisms measured in micrometers. Organism

Average

STDV

Paramecium

Size (μm) 148

13

Amoeba

258

28

Human Red Blood

10.1

0.74

Cell Table 2. Raw data of each organism measured in micrometers (μm). Paramecium

Amoeba

RBC

152

266

10

133

221

10

159

290

11

-

271

10

-

273

9

-

225

11

-

-

10

-

-

10

-

-

9

-

-

11

4 2

Figure 1. Sample paramecium

Analysis: Based off the average size in micrometers of the amoeba, paramecium, and human cell blood cells observed, the amoeba seems to be the largest, as well as has the greatest variation in size. The amoeba measured the largest average size at 258±28 μm, followed by paramecium with an average size of 148±13 μm, and lastly human red blood cells with an average size of 10.1±0.74 μm. Along with being the largest subject out of the experiment, the amoeba were also the most variable in size. They presented the largest standard deviation value, which means all of the amoebas observed did not show as much consistency in size as the paramecium and red blood cells. In contrast, the red blood cells being the smallest out of the three subjects, displayed the most consistency in measurements compared to the other two. From this data, we can infer that the largest an organism or cell is, the more inconsistent or variable the sizing ought to be.

Conclusions: Microscopy is a very versatile tool used in cell biology as there as many different techniques that can be utilized for specific specimen of interest. For example, fixed slides are useful for the observation of nonliving things, whereas wet mounts and depression slides are used for observing fresh or live organisms. Microscopy was used to measure the size of paramecium, amoeba, and human red blood cells to determine their variability in size. According to the data, amoeba were the largest and most variable subjects we studied with an average size of 258±28 μm, followed by paramecium with an average size of 148±13 μm, and lastly human red blood cells with an average size of 10.1±0.74 μm. Based on the relationship between size and variability seen from the data, it seems that the two variables are positively associated—meaning as one gets larger, the other does too.

5 2

Troubleshooting: Problem: Unclear image, hard to see subject. Solution: Check for air bubbles on the slides and carefully remove them. Problem: Image too small to see. Solution: Use a higher objective lens to zoom in more.

6 2 Lab exercise 2: Micropipette Technique Review and Spectrophotometric Methods Date: 9.16.2020 Objective: The objective of this lab is to review micropipetting techniques and etiquette, as well as demonstrate how the Beer-Lambert law coupled with spectrophotometry can be used to determine unknown protein concentrations using the absorbance data and generated standard curve. Spectrophotometry is a method used to measure how much a substance absorbs light by measuring the intensity of light as a beam of light passes through the sample solution, which can then be used to calculate the concentration. An example of what spectrophotometry is used is protein concentration determination. The absorbance measured at 280 nm and 205 nm can be used to calculate protein concentration as well as quantifying “crude lysates and purified or partially purified protein.” 1 In addition to studying protein concentrations, spectrophotometry can be used to study protein-protein interactions over a wide concentration range. This is achieved by studying the correlation between the change in absorbance measurements and viscosity as a function of protein concentration. 2 Works Cited:

1. Simonian M. H. (2002). Spectrophotometric determination of protein concentration. Current protocols in cell biology, Appendix 3, . https://doi.org/10.1002/0471143030.cba03bs15 2. Thakkar, S. V., Allegre, K. M., Joshi, S. B., Volkin, D. B., & Middaugh, C. R. (2012). An application of ultraviolet spectroscopy to study interactions in proteins solutions at high concentrations. Journal of pharmaceutical sciences, 101(9), 3051–3061. https://doi.org/10.1002/jps.23188 Pre-lab: II-A: To dispense 500 μL (0.5 mL), use a p1000 pipette, Volume window setting 050. To dispense 0.2 mL, use a p1000 or p200 pipette, Volume window setting 020 or 200. To dispense 4.8 μL, use a p20 pipette, Volume window setting 048.

II-B: 1) Bovine serum albumin (BSA) is a serum albumin protein derived from cows (usually from a fetal bovine source). BSA is separated from whole blood using a multi-step fractionation process. Dr. Edwin J. Cohn of Harvard University, found that blood proteins could be separated from each other by changing the temperature and varying concentrations of an organic solvent. This process used those variables to separate human blood plasma into five fractions--to which the fifth contains mostly albumin. This is why BSA is also referred to as "Fraction V." Source: https://www.gembio.com/post/bsa-cohn-cold-etoh-vs-

7 2 heat-shock#:~:text=How%20is%20BSA%20made%3F,a%20multi%2Dstep%20fractionation %20process.&text=His%20process%20used%20these%20two,was%20called%20%E2%80%9CFraction %20V%E2%80%9D. 2) 1. to stabilize some restriction enzymes during digestion of DNA 2. to determine the quantity of other proteins 3. is considered to be a universal blocking reagent Source: https://rockland-inc.com/bovine-serum-albumin.aspx#:~:text=BSA%20(often%20from%20a %20fetal,pipet%20tips%2C%20and%20other%20vessels. 3) BSA is extracted from the blood of cows and since cow blood is a widely available byproduct of the cattle industry, BSA is abundantly available for use. Source: https://info.gbiosciences.com/blog/why-isbovine-serum-the-preferred-standard-for-protein-assays

Materials: 

96-well plate



P-200 and P-20 micropipette



Distilled water



2 mg/mL BSA stock solution



Bradford reagent



Unknown BSA protein sample



Microplate reader

Procedures: 1. Created BSA standard serial dilutions for six different concentrations (including stock). 2. Generated triplicates of each diluted BSA standard. 3. Created serial dilutions for unknown BSA protein sample for four different concentrations (including stock). 4. Generated triplicates of each diluted unknown sample. 5. Added Bradford reagent to all of the triplicate samples including the BSA standards and unknown samples. 6. After 5-minute incubation time, inserted microwell with all the samples into microplate reader to determine the average absorbance readings of each triplicate at 595 nm.

Procedure detailed in “Part I. Protein Standard and Unknown Sample Preparations” in the BSCI 330 Lab Manual.

8 2 Cantemir-Stone, Carmen, editor. “An Introduction to the Study of Cells.” BSCI 330 Laboratory Manual: Cell Biology and Physiology - University of Maryland. Macmillan Higher Education, 2020, pp. 35-37. Results: Table 1. BSA concentration in mg/mL and average absorbance at 595 nm for Hans’s dilutions. Hans’s Dilution

Dilution Factor

Concentration BSA

Average A595

D0

1:1

1

1.027333333

D1

1:2

0.5

0.886333333

D2

1:4

0.25

0.673

D3

1:8

0.13

0.574333333

D4

1:16

0.07

0.473666667

Blank

1:0

0

0.401666667

(mg/mL)

Obj ect3

Figure 1. BSA standard curve for Hans’s dilutions; average absorbance at 595 nm against BSA concentration in mg/mL.

9 2 Table 2. The calculated protein concentration in mg/mL for Han’s unknown BSA protein sample, based off the dilution factor, standard curve, average absorbance at 595 nm. Hans’s

Dilution Unknown

Average A595

Calculated Protein Concentration (mg/mL)

Factor D1

1:2

1.872 1.633

D2

1:4

1.386 1.33123

D3

1:8

0.947 1.0586667

D4

1:16

0.621 0.89833333

Protein concentration calculations: D1: x= (1.633-0.471)/0.6206= 1.872 D2: x= (1.33123-0.471)/0.6206= 1.386 D3: x= (1.0586667-0.471)/0.6206= 0.947 D4: x= (0.89833333-0.471)/0.6206= 0.621 Stock protein concentration calculation:

0.621

mg mg ×16=9.936 mL mL

Table 3. BSA concentration in mg/mL and average absorbance at 595 nm for Brad’s dilutions. Brad’s

Dilution Factor

Dilution

Concentration BSA

Average A595

(mg/mL)

D0

1:1

1

0.625666667

D1

1:2

0.5

0.484666667

D2

1:4

0.25

0.271333333

D3

1:8

0.13

0.172666667

D4

1:16

0.07

0.073241202

Blank

1:0

0

0.00912

10 2

Obj ect7

Figure 2. BSA standard curve for Brad’s dilutions; average absorbance at 595 nm against BSA concentration in mg/mL.

Table 4. The calculated protein concentration in mg/mL for Brad’s unknown BSA protein sample, based off the dilution factor, standard curve, average absorbance at 595 nm. Brad’s

Dilution Unknown

Average A595

Calculated Protein Concentration (mg/mL)

Factor

D1

1:2

1.304188

2.000

D2

1:4

0.6892382

1.001

D3

1:8

0.3797

0.499

D4

1:16

0.2259994

0.249

Protein concentration calculations: D1: x= (1.304188-0.0726)/0.6159= 2.000 D2: x= (0.6892382-0.0726)/0.6159= 1.001 D3: x= (0.3797-0.0726)/0.6159= 0.499 D4: x= 0.2259994-0.0726)/0.6159= 0.249 Stock protein concentration calculation:

11 2

0.249

mg mg ×16=3.984 mL mL

Analysis: Utilizing the absorbance values measured at different concentrations of BSA, a BSA standard curve was plotted for both Hans and Brad. The linear equation yielded from the BSA standard curve graph for Hans was y=0.6206x-0.471, whereas the linear equation for Brad was y=0.6159x-0.0726. Based on the slope of the equation alone, one can estimate that Hans’s stock protein concentration will be larger than Brad’s. Plugging in the absorbance values measured from unknown protein concentrations to the standard curve equations, the protein concentration was determined. Given this information and the dilution factor, the stock protein concentration for Hans and Brad was calculated to be 9.936 mg/mL and 3.984 mg/mL respectively. Hans’s stock protein solution had a higher concentration than Brad’s by about 2.5 times.

Conclusions: The overall goal of the experiment was to be able to determine the protein concentration of an unknown BSA protein sample using a curated BSA standard curve. By taking the absorbance of various dilutions of a BSA sample of known concentration, a standard curve was able to be generated. With the help of this standard curve and Beer’s Law, the protein concentrations of the unknown samples were able to be calculated. Finally, the concentration of the stock unknown sample was able to be calculated by incorporating the dilution factor and estimated concentration. The calculated concentration for Hans’s unknown was found to be 9.936 mg/mL, whereas Brad’s was found to be 3.984 mg/mL. The concentration of Han’s unknown seems to be about 2.5 times, or 250%, larger than Brad’s.

Troubleshooting: Problem: Absorbance values are significantly higher than normal. Solution: Make sure to calibrate the spectrophotometer and to use a blank.

Problem: Absorbance values are significantly lower than normal. Solution: Make sure the cuvette is sufficiently filled up with the sample.

12 2 Lab exercise 3: Protein A: Properties and Methods of Isolation Date: 9.23.2020 Objective: The objective of this experiment is to understand how the chemical properties of proteins can determine their interactions with water and how this can be used to isolate proteins from tissues and cells. Methods of isolation to be studied include centrifugation, salting out, the use of an organic solvent, and SDS-PAGE—the two main protein precipitation methods being salt precipitation and organic solvent precipitation. Centrifugation is widely used to isolate and purify many biological materials like viruses and subcellular organelles. Exosomes, for an example, are membrane-bound extracellular vesicles that can also be carriers for disease markers. 1 Centrifugation of these exosomes can be valuable for biomedical and immunological research. Additionally, this method of molecule isolation can also be very beneficial in disease diagnosis as centrifugation has been utilized to quantify colorectal cancer-related mRNA in plasma.2 Works cited:

1. Livshits, M. A., Khomyakova, E., Evtushenko, E. G., Lazarev, V. N., Kulemin, N. A., Semina, S. E., Generozov, E. V., & Govorun, V. M. (2015). Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol. Scientific reports, 5, 17319. https://doi.org/10.1038/srep17319 2. Xue, Vivian Weiwen et al. “The Effect of Centrifugal Force in Quantification of Colorectal Cancer-Related mRNA in Plasma Using Targeted Sequencing.” Frontiers in genetics vol. 9 165. 15 May. 2018, doi:10.3389/fgene.2018.00165 Pre-lab: III-A: RPM stands for “revolutions per minute,” whereas RFC stands for “relative centrifugal force.” RPM is about the number of revolutions (or turns) an object is rotating per minute. RFC is about the force applied on an object while it is rotating. The RFC can be calculated using the RPM and the radius. Source: http://www.differencebetween.net/business/product-services/differences-between-an-rcf-and-anrpm/#:~:text=Summary%3A,the%20RPM%20and%20the%20radius.

13 2 III-B: Relative % of

Types

Plasma by Weight Protein

8%

Albumins, globulins, immunoglobin, fibrinogen

Water

91%

Water

Other solutes

1%

Sodium, potassium, calcium, chloride

Source: Mathew J, Sankar P, Varacallo M. Physiology, Blood Plasma. [Updated 2020 Apr 25]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK531504/ Leeman, M., Choi, J., Hansson, S., Storm, M. U., & Nilsson, L. (2018). Proteins and antibodies in serum, plasma, and whole blood-size characterization using asymmetrical flow field-flow fractionation (AF4). Analytica...