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A STUDY ON THE ISOLATION & ANALYSIS OF NUCLEIC ACIDS

Posted by DR. NURVUS on April 6, 2016
Posted in: Biochemistry. Tagged: Biochemistry, biology, cell, chem431, Chemistry, DNA, electrophoresis, experiment, hyperchromic, lab, lab report, nucleic acid, RNA, spectroscopy, USI. Leave a comment

cropped-dna-163466_1280.jpg

A Study on the Isolation & Analysis of Nucleic Acids

Basis in Isolation, Ultraviolet Spectroscopy, Restriction Endonuclease Digestions, Ligations, Hyperchromic Effect, Florescence Spectroscopy, and Agarose Gel Electrophoresis

 

Abstract

The quantitative analysis of nucleic acid samples can offer up insights into samples with unknown properties. In this brief study multiple techniques are utilized to isolate, quantify, and discern nucleic acid residues from Escherichia coli (E. coli) plasmids. Restriction digests and hyperchromic assays were also developed. Results for the restriction digest were not conclusive, but the hyperchromic effect was observed in both RNA and DNA as expected. The denaturant study with NaOH saw an interesting result, with RNA showing little change in spectroscopic absorbance, while DNA showed a drastic increase. Continue Reading

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A STUDY ON ENZYMES AND CATALYTIC ACTIVITY

Posted by DR. NURVUS on April 6, 2016
Posted in: Biochemistry. Tagged: Biochemistry, biology, catalysis, catalytic, cell, chem431, Chemistry, enzymes, hemoglobin, horseradish, lab report, myoglobin, peroxidase, USI. Leave a comment

cropped-cropped-acetaldehydedehydrogenase-1nvm.png

A Study on Proteins, Enzymes, & Catalytic Activity

Basis in Column Chromatography, Enzyme Activity Assay, Protein Assay, Gel Filtration, and Enzyme Assay

Abstract

Proteins are the building blocks of our bodies and many fall into the category of enzymes which play critical roles in the biochemical reactions which keep us alive. Understanding enzymes, and proteins in general, is crucial to our overall understanding of the biochemical make-up of our internal systems, as well as those of Earth’s other organisms. Horseradish peroxidase (HrP) is an example of an enzyme that is sometimes utilized in biochemistry for its economic benefits and ease of isolation. In this experiment, column and gel-filtration chromatography were used to isolate HrP. Enzyme assays were then used to determine the catalytic activity of the isolate. Determination of the molecular weight was made through gel electrophoresis. These techniques were shown to be viable and are crucial for proper protein analysis, and yielded a better understanding of HrP’s catalytic activity.


Introduction
Proteins are types of macromolecules consisting of chains of amino acid residues.
Protein production begins with transcription of mRNA within the nucleus and ends within the confines of the rough endoplasmic reticulum and golgi apparatus. Once transcription is complete and the spliced mRNA is chaperoned out of the nucleus through the nuclear pores, ribosomal subunits come together and begin translation of the mRNA, beginning at the start codon and reading along until terminated. Every three nucleotides forms a codon which within the ribosome allows the attraction and chemical reactions required to develop a polypeptide. Amino acid residues make each unit of the polypeptide up, linked
via their peptide bonds, and they can be substantially large for some proteins (Voet et al., 2013). Once translation is complete the protein must be properly folded into its correct figure01shape so that it may function properly and meet the cell’s needs. Often the proteins created within the cell are enzymes, or proteins which catalyze chemical reactions (Klug et al., 2009). Enzymes drive the metabolic process and without the work they do, lowering activation energy required for reactions to progress, we would not be able to function.

Proteins typically have very specific functions in the body. For instance the two proteins, Myoglobin and Hemoglobin, both bind oxygen, but they have vastly different roles. Myoglobin is found within the tissues of the muscles, binding oxygen for muscle cells. It contains an iron-heme pigment group which gives our muscles their red coloration (Voet et al., 2013). Interestingly, myoglobin was the first protein to be three-dimensionally determined using X-ray crystallography, achieved by John Kendrew and associates in 1958 (NSF, 2004). Hemoglobin, in contrast to myoglobin is much more complex, essentially containing four myoglobin-like subunits, two α-globin and two β-globin, thereby allowing one hemoglobin to carry four oxygen molecules. (Alberts et al., 2008) hemoglobin is responsible for carrying oxygen from the lungs to the peripheral tissues for almost all vertebrates.

In this experiment we look at horseradish peroxidase and myoglobin specifically.figure 2 HrP also contains a heme pigment group with an Iron atom like myoglobin and hemoglobin. Therefore it is a metalloenzyme as well, on top of being a glycoprotein (Collins, 2014). The heme group cofactor seen in these enzymes is depicted by figure 1, showing the binding of a molecule of oxygen and some of the resonance which occurs to facilitate this binding.

Horseradish peroxidase is found in the roots of horseradish plants (Armoracia rusticana). Its overall structure is mostly alpha helical as depicted by figure 2 (Constantino et al., 2010), with some irregular loops and the heme cofactor at its center.

Myoglobin, as stated previously is a monomeric enzyme which contains a “heme prosthetic group” (Voet et al., 2013) and falls into the globin family of proteins. The ring system which makes up heme is heterocyclic in structure, and contains four pyrrole groups with methene linkages. Myoglobin’s iron oxidizes to Fe(III) from Fe(II) forming metmyoglobin or methemoglobin, which gives old meat and dried blood their brownish colorings. Just like in hemoglobin, other small molecules beside oxygen can bind to myoglobin’s heme rings, such as carbon monoxide, nitrogen oxide, and dihydrogen sulfide, many of which have a greater affinity for the binding site then oxygen. This causes the active site which makes this enzyme useful to become blocked. (Voet et al., 2013)

Proteins in general can be purified or isolated using multiple techniques such as centrifugation, electrophoresis, as well as a multitude of different chromatography techniques. Other tests typically conducted on proteins for determination of activity and structure include x-ray crystallography, nuclear magnetic resonance, and mass spectrometry, to name a few.

 


Materials & Methods

The procedure and materials used were in following with the published procedure (Collins, 2014).


Results

Affinity Column

Kinetic data plotted for determination of enzyme activity in absorbency units/time. Table 1 refers to all data obtained via each fraction from the affinity column which was eluted and read. Figure 3 depicts this data showing the slope rate vs fraction number, in essence the slope of the absorbency units/time, in this case giving a visual depiction of enzyme activity.

fig3

fig4

Figure 4 shows a chromatography profile graph derived from the affinity column data in the fractional table. Both the readings at 280 & 403nm were plotted. Enzyme activity in absorbance units over time was plotted against the fraction number. Table 2 depicts the calculated data for the crude, pellet, supernatant, and pooled fractions samples. Each sample volume was a ten microliter sample which was converted from the slope/kinetic data into the enzyme activity/sec/mL.

fig4-1

table2 (4)

The graph below depicts the enzyme assays by fraction against their absorbance. This is a linear plot depicting enzyme activity. Fractions 12 and 16 are not shown because they did not have any activity in their samples.

fig5

Cary 300 UV/VIS absorbance fraction graph for the fraction sets in the UV/VIS spectrometer. Fractions show relative absorbance for each sample measured.

fig6

Gel Filtration Columns

This graph, Figure 7, depicts the log M.W. of standards vs their Kd values, using this standard it is possible to evaluate myoglobin and peroxidases molecular weight. Myoglobin is approximately 17 Kda, while Peroxidase is approximately 44 Kda (main peptide chain minus sugar and calcium/heme is 34Kda.

fig7

This graph, Figure 8, depicts the BSA standard ran at 595nm on the Milton Roy UV/VIS spectrometer.

fig 8

The following graphs, Figure 9 and 10, depict the Gel Filtration Column data for Peroxidase and Myoglobin.

fig 9

fig 10

SDS-PAGE

Here, Table 3 depicts the electrophoresis protein standards, giving the relative mobility (Rm) for each protein sample that was ran in a lane. Figure 11 is a graph showing the log molecular weight versus the Rm.

 table3

fig11

Table 4 gives the molecular masses of each protein as determined by gel filtration, SDS, and the current scientific literature.

table4


Discussion

This experiment highlighted multiple lab techniques crucial to working with proteins. Enzyme activity and purity were determined following gel filtration. For the affinity chromatography, concanavalin-A, a lectin, was used in the column as it binds to peroxidase readily via binding to the carbohydrate side chains (since it’s a glycoprotein), which allows everything else to flush through the column. Once the rest is through, the use of mannose, which concanavalin-A has a higher affinity for, is utilized to wash the peroxidase back into the matrix for collection. Another type of lectin, such as lentil lectin or snowdrop lectin could be used, which also have an affinity for mannose.

The fold purification seen in table 2 shows how much the affinity column purified the sample, where the crude is at 1, since the samples specific activity is divided by its own specific activity. This is because this sample contains all the peroxidase possible. The supernate had a fold of 10.6, the re-suspended pellet had a fold of 2.0, and the pool(13+14) had a fold of 10.8, slightly higher than the supernate. The supernate here should indeed have a higher specific activity and if the gel column does well, higher fold. This is seen, and should also be expected in the pool as the pool was selected to highlight the most probable elutions with the highest percentage of peroxidase. The pellet after being re-suspended following the pour off of the supernate should contain little peroxidase, yielding a lower specific activity and a lower fold, which is what is observed. Yields for the supernatant and pool were high, with crude being at 100% as expected. The pellet showed a lower yield which is also expected. Yields over 100% shouldn’t be observed but this could be attributed for high concentrations of peroxidase in low volume solutions and improper calculations attributed to mis-use of proper units.

The molecular weight of myoglobin in the literature is 17Kda, with peroxidase molecular weight being (total) 44Kda. The peroxidase peptide chain alone is ~34Kda. The 44Kda literature value includes the peptide chains, heme, calcium, and carbohydrate groups. Gel filtration, allowed using the Kd vs log of m.w. showed lower Myoglobin, which was also seen in the SDS-PAGE from the electrophoresis with a m.w. of 6.3Kda, which is substantially lower than the lit value of 17.0Kda. This could be due to discrepancies during the development of the myoglobin solution or there could actually be two, hard to distinguish bands in the same area, which would give about 14Kda which is closer to the lit value. Peroxidase did well, with experimental values of 57Kda total from the SDS-PAGE, which can most likely be attributed to additives and additional residues left over in the solution, which can be seen to still be associated with the pellet assay. Subtracting these out would yield a more reasonable number around the literature value.

Visually, the enzymes obtained were not very pure. The dying that occurred was very hard to discern immediately following removal of the gel from the dye solution. The yield % therefore is incorrect, but the fold calculation is more discernible and shows the overall purity of the sample well. This estimation is reasonable in the sense that if there is little enzyme in the wells then there will be less enzyme for the dyes to adhere to, which correlates to an image that is harder to work with. That being said, the peroxidase was most likely not pure for the fact that the molecular weight data also showed it having a higher overall Kda than the literature values. Reasonably, this can be assigned to impurities in the sample.


References

  1. Collins, J., Proteins and Catalytic Activity,Biochemistry I Laboratory Experiments [Online] 2014, 13th edition. Blackboard. http://blackboard.usi.edu/ (November 24, 2014).
  2. Voet, Donald., Voet, J. G., Pratt, C. W., Fundamentals of Biochemistry, 4th 2013. p93-354.
  3. NSF, (U.S.) National Science Foundation: Protein Data Bank Chronology [Online], 2004.   http://www.nsf.gov/news/news_summ.jsp?cntn_id=100689 (Jan. 21, 2004).
  4. Constantino, Z., Amedeo, P., Andrea, A. “On the catalytic role of structural fluctuations in enzyme reactions: computational evidence on the formation of compound 0 in horseradish peroxidase”. Faraday Discuss, 2010. 145,107-119, DOI: 10.1039
  5. Klug, W., Cummings, M., Spencer, C., Palladino, M., Concepts of Genetics, 9th 2009. p6-9.
  6. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Waleter, P., Molecular Biology of the Cell, 5th 2008. p124-193.

Photos

polyacrylamide gel set-up 02
polyacrylamide gel set-up 02
gel_UV
gel_UV
acrylamide protein gel electrophoresis
acrylamide protein gel electrophoresis
enzyme catalytic assay
enzyme catalytic assay
polyacrylamide gel set-up 01
polyacrylamide gel set-up 01
affinity column, fractions
affinity column, fractions

Date: 24 November 2014
CHEM431.002

STEM Specific Search Engines, MetaCrawlers & More

Posted by DR. NURVUS on April 6, 2016
Posted in: Free Resources. Tagged: biology, Chemistry, citation, citations, directory, grad students, graduate students, help, lab resources, lab spend, lab start tips, metacrawler, metasearch, PhD, post-doc, science, Science/R&D, search, search engine, starting out, starting your lab, STEM, tips to starting lab, web tools. Leave a comment

mag02

Sometimes scouring the vastness of the internet can be a hassle. You don’t have time to sift through the millions of database and journal sources for your research paper, proposal, or summary. You already have 10^30 things you need to do.

Well I went ahead and did some of that scouring for you. Hopefully these database driven search engines are useful and can provide you with a portal to the articles you need to keep up on your to-do list.

Useful STEM-base Search Engines

  1. Google Scholar
  2. SciFinder 
  3. Wiley Online Library
  4. ScienceDirect
  5. RefSeek
  6. Lab Spend
  7. CiteSeerx
  8. GetCITED
  9. Microsoft Academic Research
  10. Bioline International
  11. Directory of Open Access Journals
  12. PLOSone
  13. BioOne
  14. New Journal of Physics
  15. NCBI
  16. Sigma Aldrich – For reagent/chemical properties

The above academic directory powered SE’s are great for scientific research and paper citations. They offer information covering almost all major areas of science including computer and technology based fields.

With that said, search engines are not the only way to sift through the information online. Though more current and comprehensive in their scope,. search engines are merely the newest most “mainstream” players on the field. Other reliable sources of information are found in large web directories which allow you to browse among dozens or hundreds of sites devoted to the same subject. The problem with directories is that they do not auto-index like Google and other search engines do, which means it may take several months for new sites to be added to their catalog.

You can also use a web-tool known as a metacrawler or metasearch site. These sites combine the work of multiple search engines and can search billions of web pages. The only drawback is that using this search option can sometimes be inferior to multiple direct search engine queries.

The last one I know of is the all-in-one search pages which, unlike metasearch/crawlers do not send your query through multiple engines, but instead list a wide variety of search engines on one page, allowing you to search without going directly to a specific search engine.

For more information and updates check out Search Engine Watch. They are a good source on the current climate surrounding the most used search tools on the web.

Web Directories

  1. Science Web Directories

MetaCrawlers & MetaSearch

  1. Dogpile
  2. Vivisimo
  3. Kartoo
  4. Mamma
  5. SurfWax
  6. Clusty
  7. CurryGuide
  8. Excite
  9. Fazzle
  10. Gimenei
  11. IceRocket – Meta search engine with thumbnail displays.
  12. Info.com – Provides results from 14 search engines and pay-per-click directories.
  13. InfoGrid – Provides direct links to major search sites and topical web sites in different categories. Meta search and news searching is also offered.
  14. Infonetware
  15. Ixquick
  16. iZito – Has a “park” feature, allowing you to essentially save specific pages from the result listings.
  17. Jux2 – This is a search result comparison tool, allowing for side by side result comparisons of multiple SEs.
  18. Meceoo – Allows you to create an “exclusion list” to block pages from particular web sites being included.
  19. MetaCrawler – One of the oldest meta search services, MetaCrawler began in July 1995 at the University of Washington.
  20. MetaEureka – Offers a nice option to see Alexa info about pages that are listed.
  21. ProFusion – Accesses several major web SEs and some invisible web resources.
  22. Query Server
  23. Turbo10 – Accesses traditional & some invisible web databases.
  24. Search.com
  25. Ujiko – Flash is required.
  26. WebCrawler
  27. ZapMeta

All-In-One Pages

  1. CentPage 
  2. Google Versus Yahoo Tool – Exactly what you think it is.
  3. One Page MultiSearch Engines
  4. Proteus
  5. Queryster
  6. YurNet

 


 

Additionally, exiting the realm of scientific discovering and entering the realm of regular discovery, there are many specialized metasearch services available which can make our life on the net a little easier.

Specific MetaSearch Services

  1. GoFish – Searches licensed and commercially available digital media downloads including music, movies, music videos, ringtones, mobile games and PC games.
  2. Searchy.co.uk – Searches 15 U.K. only search engines.
  3. Watson – for macintosh users.

If you are still having trouble with all the internets setting there before you, then it may be beneficial for you to read through the Science on the Internet site. It is a tutorial developed by the International Network for the Availability for Science Publications (INASP) and the Institute for Learning and Research Technology (ILRT) at the University of Bristol to help policy advisers who want to use the Internet to find and manage information on science topics.

**ProTip – Always check your university database as they have access to many journals and if the article you are looking for is not in one of them you can request an inter-library loan. A word of warning, this can sometimes take a couple days so plan in advance.


The above is by no means everything, but hopefully if you are studying in the STEM fields it can provide you with some helpful sources. As always, leave me a comment if you find any sites that belong here and especially if any of the links are dead!

Remember unless science is accessible to all, it cannot be as beneficial for all.

 

 

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