BIOL2030 Lab 4: Fluorescent Protein Analysis Guide

Biol 2030U – Fall 2023 Analysis of results Laboratory Assignment # 4 Your Name: ID #: CRN #: TA’s name: Make sure to bring your laptop to this lab session. You won’t be allowed in the lab otherwise. Pre-lab assignment: Make sure you complete the Pre-lab assignment before coming to the lab and submit a hardcopy to your TA at the beginning of the lab. If the questions have not been answered, you will not be allowed to attend the lab. Make sure you bring a printout of your Assignment file (you can print both-sided). You won’t be allowed to attend the lab without this document’s printout. Staple the pages of your Assignment, so you don’t lose any pages. Note: for this lab, you do not need to print the Intro/protocol file. For this laboratory assignment, perform the calculations, fill in the tables, prepare the figures and answer the questions. Make sure you write or type your answers in your own words . Write/type in the space provided (the line numbers are indicated). You will work individually in this lab. Make sure everything you are submitting is done on your own. Your lab assignment ( pages 1 to 6 ) is due at the end of your lab session via Canvas dropbox Please show your submission confirmation to your TA before leaving the lab. Late submission won’t be accepted. Only people who attend the full lab can submit an assignment for correction. ● Do not submit pages 7 to 9 as you will need them to complete your write-up. ● Make sure you have pages 7 to 9 initialized by your TA for completion before leaving. Failure to do so will result in a -10% penalty on your results and Discussion assignment. 1 | Page

General questions (you can answer these questions before coming to the lab): You have run both non-denaturing (ND) and denaturing (D) samples of your purified fluorescent proteins on the SDS-PAGE gel. In which case(s) do you expect to see a different band pattern between the two samples (Y for yes pr N for no)? Briefly explain. Protein conformation Diff bands ND / D (Y or N) Explain Monomer No Running its native and denatured form separates the protein into its polypeptides however a monomer only consists of one polypeptide so both forms would indicate the same molecular weight as one band Dimer Yes Running the native and denatured form will separate the protein into its polypeptides. Since a dimer consists of two polypeptides, the denatured form will indicate a band pattern for each polypeptide with a different molecular weight from the native form which will indicate the molecular weight of the two polypeptides as a unit and only form a different band pattern. Tandem dimer no Tandem dimers often express a monomer behaviour when denatured. In a tandem dimer, two identical units are arranged and bonded by a noncovalent bond. Both the denatured and natured form will produce a similar band pattern Tetramer Yes Running the native and denatured form will give us different band patterns. A tetramer is a protein with 4 polypeptide subunits. The subunits will separate into its individual subunits when denatured and give us a different band pattern than its native form 2 | Page

which would indicate the entire protein as a unit. What is the number of amino acids and the molecular weight of the in the GFP protein and DsRed monomers? (Include your source/reference) GFP protein: 27 kDa Source/reference: https://bmcbiotechnol.biomedcentral.com/articles/10.1186/s12896-019-0590-y DsRed protein: 32 kDa Source/reference: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC122988/ 3 | Page

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

1. Spectral analysis of your sample Draw the spectral curve (Figure 1) using Excel and record the wavelength showing maximum absorption: 554-556nm Figure # and legend- Figure 1: The spectral curve of absorbance of the unknown fluorescent protein at wavelengths varying from 400-700 nm with a 2 nm increase between measurements. The unknown protein pSECT-FP12 was transformed to competent E-Coli JM109-DE3 and then isolated and purified before the absorbance values were obtained using a spectrophotometer. Does this absorption correlate with one of the excitation maximum reported in Table 1 (see introduction)? Otherwise, can you narrow down the possible proteins your unknown might be? Briefly explain. Our excitation value directly correlates with the excitation maximum of 554 nm reported in Table 1. for dTomato which is a dimer and tdTomato which is a tandem dimer. It can be assumed that our unknown protein is one of these two fluorescent proteins. DsRed which is a tetramer also has a relatively close excitation maximum of 558. considering our excitation ranged from 554-556, this is only 2 nm away from the value of 558, So it is important to consider this protein has a possibility of being our unknown fluorescent protein. 2. SDS-PAGE 4 | Page

Your gel samples: _______________________ (indicate the gel number and lines where your samples were loaded) Figure # and legend- Figure 2 The polyacrylamide gel electrophoresis results on an SDS-page with respective band patterns for the crude lysate, non denatured unknown fluorescent protein and denatured unknown fluorescent protein. The column furthest left represents the Molecular weight marker. The crude lysate was isolated from other cytoplasmic proteins. The native proteins were isolated and purified using affinity chromatography. The denatured proteins were isolated, purified and denatured in a hot water bath. - Comment on the efficiency of the Ni-NTA purification technique (comparing CL to E1)? When comparing the crude lysate to the purified proteins, it can be observed that many of the unclear bands were removed during purification. However it can be. noted that a few of the unclear bands remain in the purified proteins. This indicates a successful purification, with the majority of the contaminants removed - Do you see difference between denaturing and non-denaturing E1 sample? How can you explain the results observed? Add any other comments. There is a clear difference in the number of clear bands between the natured and denatured protein samples. The native protein shows two distinct bands indicating a tandem dimer that has two polypeptides covalently bonded together. The denatured form will act as a monomer and 5 | Page

travel as a single unit giving us a single band. This is because the two covalently bonded polypeptides are identical. - Looking at both the results of the spectral analysis and the SDS-PAGE gel data, can you identify your unknown protein? Explain your reasoning. Knowing the values of our excitation maximum, we can conclude our protein is likely either a dTomato or a tdTomato while still considering the possibility of DsRed. We can conclude we are working with a tandem dimer based on our results from the SDS-Page gel data. With this information, it is evident that we are working with tdTomato as it falls within the excitation maximum of our protein and follows the correct band patterns in the SDS-page. Analysis of the gel picture given to you Now, analyze the three proteins samples (each have 3 lines: CL; E1-ND and E1-D) using the gel picture provided. This analysis is the one you will use to write your Results and Discussion assignment. Table 2: Molecular weight and migration distance of the marker on the SDS-PAGE Column 1 Column 2 *1 Column 3 *2 MW (kDalton) Log MW Distance of migration (cm) 116 2.064457989 2.6 cm 66.2 1.820857989 4.30 cm 45 1.653212514 6.0 cm 35 1.544068044 7.0 cm 25 1.397940009 8.20 cm 18.4 1.264817823 8.80 cm 14.4 1.158362492 9.10 cm *1 Calculate the Log MW from the values in Column 1 *2 Use the picture to measure the migration distance of each band (from bottom of the wells to bottom of the band) using a ruler. Make sure you identify each band correctly on the gel. Draw the standard curve (Figure 2) using Excel. The standard curve is made by plotting Log MW (on Y-axis) versus migration distance (X-axis). Note that the Log MW does not have units. Draw the best fit line (must be straight) and determine the equation for the line. 6 | Page

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

Figure # and legend- Figure 3: The standard curve of the log of molecular weight plotted against the distance of migration observed for the molecular weight markers on the gel electrophoresis SDS-Page. A negative linear relationship between the two values can be observed. Submit your lab report (up to and including this page) by the end of your lab period in the Canvas dropbox. Analysis of the 3 unknown proteins on the SDS gel TAKE the NEXT THREE PAGES WITH YOU; make you’re your TA initializes the pages before leaving the lab. Only measure the migration distance of the bands with a red dot in all three sample sets and use the standard curve to determine the corresponding molecular weight of the bands in all sets of three samples. Record your results in Table 3. 7 | Page

Table 3: Excitation Maximal absorbance, distance of migration and molecular weight of the main protein bands from the CL, E1-ND and E1-D samples Excitation Max Distance of migration (cm) Log MW MW (kDalton) CL-set 1 _______ 554 nm 7.2 1.09062 12.32026 E1-ND-set 1 5.8 & 7.2 1.29488 19.71878 E1-D-set 1 7.1 1.10521 12.74119 CL-set 2 _______ 554 nm 4.5 1.48455 30.51757 E1-ND-set 2 4.4 1.49914 31.56022 E1-D-set 2 4.5 1.48455 30.51757355 CL-set 3 ________ 548 nm 7.0 1.1198 13.17649799 E1-ND-set 3 7.0 1.1198 13.17649799 E1-D-set 3 7.3 1.07603 11.91324299 Only determine the molecular weight of the bands with a dot. TA’s initials: _______________ 8 | Page

Comment on the results obtained. Was the Affinity Chromatography technique efficient in isolating and purifying the proteins of interest? When comparing the crude lysate to the 3 purified proteins, many of unclear bands were removed during the purification process. However, some of the unclear bands remain in the purified protein samples. We can conclude that we had a successful purification, with the majority of the contaminants removed, however not all contaminants were removed. Can you identify the three unknown proteins? Explain how you came to your conclusion using both the spectral data and the gel analysis. Sample 1: Using the spectral analysis data, we concluded this protein to be either dTomato or tdTomato based on the value of its excitation maximum. The SDS-page determined the conformation of the protein to be a tandem dimer. Based on the spectral analysis data and the conformation of the protein, it can be concluded that sample 1 used tdTomato. Sample 2: Using the spectral analysis data, we can conclude using table 1, that the possible proteins with a corresponding excitation maximum would be tdTomato and dTomato. The band patterns observed on the SDS-page do not indicate a tandem dimer like observed in the first sample. The bands represent a dimer protein with two polypeptides. Using the data from spectral analysis and the SDS-page along with a process of elimination, it can be concluded that sample 2 used dTomato Sample 3: Based on the excitation maximum from spectral analysis for this sample, we can predict that the protein being used is a mOrange fluorescent protein as the excitation maximum values correspond with one another. When looking at the results from the SDS-page, we can see that there is no change in the band pattern between the denatured and non-denatured forms. This indicates we are working with a monomer which further indicates the use of mOrange. TA’s initials: _______________ 9 | Page

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

Results section: Which Figures/Tables are you planning to use in the Results Section of your report? Write the titles of each Figure and/or Table: Which subtitles are you planning to use for your Results section? Which Results will you cover in the Discussion section? TA’s initials: _______________ 10 | Page

11 | Page

BIOL2030 -Lab 4 - Lab Assignment - Fall 2023

Related Documents