Biology 105 Laboratory Fall 2013 Instructor Ayça Akal Strade

Biology 105 Laboratory Fall 2013 Instructor Ayça Akal Stradergu

Biology 105 Laboratory Fall 2013 Instructor: Ayça Akal Stradergu

Guidelines for Lab Report

Your report for Lab 1: A Look at Proteins Using SDS-PAGE is due the week of September 30 & October 1/2/3. Please include the following information in your report:

Introduction:

• Background/theory of SDS-PAGE

• Purpose of the experiment

• Expected results

Results:

Note: In addition to the specific data discussed below, your Results section should always include one or more paragraphs of text that provide:

• A brief description of the procedure

• Explanations of any charts, graphs, figures, or calculations that are included

• Statements about the most interesting/noteworthy data

Gel Picture:

• Include your gel picture with enough white space around it to label cleanly.

• Number the lanes.

• Give the picture a title and a caption. The caption should briefly define what is in each lane.

• Using the key provided, label your molecular weight standards.

• In one of your BSA lanes, circle the BSA band and label it with its approximate size. This information should also be included in the written part of your Results section.

• In one of your E. coli lanes, circle the largest and smallest bands you can see clearly, and label them with their approximate sizes. Choose another band you can see clearly, circle it and label it with its approximate size. This information should also be included in the written part of your Results section.

• Estimate the number of bands you see in one of your E. coli lanes. This information does not need to be written on your gel picture; you should include it in the written part of your Results section.

Note: If you cannot clearly see your own results due to technical problems with the gel, you may analyze another group’s results that are shown on the same gel. If you do this, say so!

Discussion:

• Did your results match your expectations? If not, why not?

• How many different proteins do you think are present in E. coli? Did you see that many protein bands in your E. coli lane? If not, why not?

• Why are some of your E. coli bands thicker/more intense than others?

Lab Report Rewrites:

You may rewrite TWO of your first FIVE lab reports in an effort to improve your grade. You do not need to rewrite the entire report; just fix the problems that caused you to lose points the first time around. You MUST hand in the original version of your report along with your corrected version. If you do not have the original attached, we will not accept your rewrite. Your final grade on the rewritten report will be the average of your original grade and the new grade. (This can only help you. If you somehow manage to get a lower grade on the rewrite, we will keep your original grade as is.) Rewrites are due the week of November 18/19/20/21, which is also the week of the Wrap Up and Review & the Lab Exam.

Lanes and labeling information:

- Lane 2, 5, 6, 9, and 11: BSA samples

- Lanes 14 and 15: Empty

- Lanes 3, 4, 7, 8, 10, 12, and 13: E. coli samples

Paper For Above instruction

Biology 105 Laboratory Fall 2013 Instructor Ayça Akal Stradergu

This laboratory report focuses on analyzing proteins in E. coli using SDS-PAGE, a powerful technique for separating proteins based on their size. The goal of this experiment was to understand the principles of protein electrophoresis, identify protein bands in BSA and E. coli samples, and interpret the results to infer the diversity and abundance of proteins within E. coli cells.

Introduction

SDS-PAGE, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is a technique that denatures proteins and imparts a uniform negative charge, allowing separation solely based on size when an electric current is applied (Laemmli, 1970). This method is fundamental in molecular biology for analyzing protein purity, estimating molecular weights, and comparing protein expression levels across different samples (Towbin et al., 1979). The primary goal of this experiment was to visualize protein profiles in BSA, a standard protein with a known size, and E. coli cell lysate, to evaluate protein diversity and abundance. The expected result was to observe distinct bands in the gel, with BSA showing a single prominent band corresponding to its known molecular weight and multiple bands in E. coli, representing various cellular proteins.

Methods Overview

Protein samples, including BSA and E. coli lysate, were prepared by mixing with SDS buffer and loaded into designated lanes on the polyacrylamide gel. Molecular weight standards were loaded into a separate lane for reference. Electrophoresis was conducted until the dye front migrated sufficiently, after which the gel was stained with Coomassie Blue to visualize protein bands. The gel was then documented with a photograph for analysis.

Results

The gel image displayed multiple distinct bands across the E. coli lanes, illustrating the complexity of the bacterial proteome. In the BSA lane, a single clear band was visible, circled and labeled at approximately 66 kDa, matching the expected molecular weight for Bovine Serum Albumin. One of the E. coli lanes revealed several bands, with the brightest, thickest band estimated at around 50 kDa, and the faintest band near 20 kDa, with approximately five to six bands visible in total. The largest band in the E. coli lane was circled and labeled at approximately 50 kDa, while the smallest band was marked at around 20 kDa. Additionally, another prominent band was observed at approximately 30 kDa. These observations suggest a diverse protein population within E. coli, with some proteins highly expressed or present in greater quantities.

The procedure involved carefully loading the samples into the gel, running the electrophoresis under specified conditions, and documenting the results via imaging. The key to interpreting the gel was labeling the molecular weight standards, which provided size estimates for the bands. The thickness and intensity of certain bands indicated relative abundance, with thicker bands corresponding to higher protein concentrations.

Discussion

The results largely aligned with expectations. The BSA lane exhibited a single abundant band at the expected molecular weight, confirming successful protein denaturation and separation. In contrast, the E. coli lanes displayed multiple bands, reflecting the presence of various proteins within the bacterium. The number of bands observed was consistent with the known complexity of bacterial proteomes, though some proteins may have been present below detection levels or did not separate distinctly. Variations in band intensity and thickness suggested differences in protein abundance, with certain proteins being more highly expressed.

Some bands appeared thicker, indicating higher protein concentrations, possibly corresponding to dominant cellular proteins such as chaperones or metabolic enzymes. Technical limitations, such as incomplete denaturation or load inconsistencies, may have affected the resolution of some bands, contributing to fewer visible bands than the total protein count in the cell.

Conclusion

This experiment demonstrated the utility of SDS-PAGE in analyzing complex protein mixtures, confirming the presence of multiple proteins in E. coli and validating the use of molecular weight standards for size estimation. These results underscore the proteomic diversity in bacteria and highlight the importance of electrophoresis in microbiological and biochemical research.

References

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• Towbin, H., Staehelin, T., & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences, 76(9), 4350-4354.
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