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Epifluorescence microscopy techniques have revolutionized modern cell and molecular biology. A) You have identified a new protein and you want to know where in the cell the protein is localized, as well as whether the protein interacts with other known signaling molecules. How would you use fluorescence microscopy to show the subcellular location of the protein? What reagents would you need? B) You suspect the protein is a motor protein that interacts with microtubules. How would you determine if this hypothesis is correct using microscopy?

Please answer every part. Thank you.

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Irving Heathcote
Irving HeathcoteLv2
29 Sep 2019
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Related questions

The overall goal of the research project that we carried out in Experimental Biology is insertion of GFP into target lapA/EF-4 gene.

1. What is the overall goal of the research project that we carried out in Experimental Biology, and how does the experiment aimed at purification of your fusion protein fit in with the overall goal of our research? How does the subcellular localization experiment fit into the overall goal?

2. What method did we use to determine whether your fusion protein was a soluble protein or a membrane bound protein? What positive control did we perform? What result was obtained from the positive control? Was this expected? Explain.

3. Know the methods used for protein purification in general as described in class, and in the file; “protein purification.ppt”

4. What method did we use to attempt to purify your LepA/EF 4 – GFP fusion protein? Why did we use this method, what advantages are there compared to other protein purification methods?

5. What is “Protein A” and how did we use it?

6. What antibody did we use in our purification experiment and why did we use it? Why can’t we use the anti-EF 4/LepA antibody to purify your fusion protein?

DAY 18, NOVEMBER 7: INTRODUCTION TO IN VIVO FUNCTIONAL ASSAYS; ASSAY FOR EXPRESSION TOXICITY AT 30°C VS. 37°C

Purpose: Compare the growth of pPEM109, pIN III, and candidate plasmids in DH5alpha-F’lacIQ -/+ IPTG to compare toxicity of functional LepA/EF-4 (expressed from pPEM109) to empty vector control (pIN III), and EF-4-GFP fusion proteins.

Background: This assay is based on the observation that E. coli is extremely sensitive to elevated levels of LepA/EF-4 expression. In prior work it was demonstrated that LepA/EF-4 expression from pPEM109 is lethal to cells growing in the presence of IPTG [Yaskowiak and March, 1995). Furthermore, pPEM109 can only be maintained in strains that contain the mutated Lac repressor gene, lacIQ, which increases repression of the operon. The wild-type promoter of the lacI gene is rather weak, resulting in ~10 molecules of Lac repressor protein per wild-type cell. The lacIQ gene contains a “promoter-up” mutation that increases transcription and Lac repressor expression 10-fold, resulting in ~100 repressor protein molecules per wild-type cell (Müller-Hill et al., 1998). Strains that only have normal Lac repressor have a slightly elevated level of “leaky” expression from the lac promoter. It was shown by comparing two identical strains (one contained lacIQ and one contained lacI) that pPEM109 could only be maintained in the lacIQ containing strain.

If you consider the structure of LepA/EF-4, and its proposed function, why do you think it would be toxic if expressed at high levels levels? What kind of mutations in the lepA gene could give rise to a mutated protein that is not toxic when expressed? What do you predict for your LepA/EF-4 - GFP fusion protein, will it be toxic when expressed or not?

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DAY 19, NOVEMBER 9: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (Lysis and Ultracentrifugation).

Background: LepA/EF-4 was originally found in the cytoplasm, membrane and periplasm when the protein was expressed from a plasmid [March and Inouye (1985) J. Biol. Chem. 260: 7206-7213]. The endogenous protein, expressed from the chromosomal gene in the absence of plasmid expression was located in the membrane [March and Inouye (1985) Proc Natl Acad Sci, USA Vol. 82: 7500-7504]. More recently it has been shown that the cellular location of LepA/EF-4 depends on the Mg2+ stress status of the cell [Pech et al. (2011) Proc Natl Acad Sci, USA Vol. 108: 3199-3203].

Here we will examine the comparative distribution of LepA/EF-4 and LepA/EF-4-GFP in cells by subcellular fractionation of native cellular protein extracts. Do you predict that your EF-4-GFP fusion protein will be localized to the cytoplasm, membrane, or both?

In contrast to our previous denaturing extract preparation, the native preparation maintains the overall shape/folding of proteins so they can localize appropriately prior to analysis. Looking ahead, how will the preparation be different in native vs. denaturing extracts?

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DAY 20, NOVEMBER 14: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (SDS PAGE and Transfer)

Procedure:

SDS PAGE Analysis

1. Each section should have the following control samples to analyze from DAY 19:

Extract Fraction Treatment

pPEM109 Input +IPTG

pPEM109 Sol +IPTG

pPEM109 Mem +IPTG

In addition, each student should have 3 experimental samples for their candidate:

Extract Fraction Treatment

Candidate Input +IPTG

Candidate Sol +IPTG

Candidate Mem +IPTG

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DAY 21, NOVEMBER 16: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (Western Blot detection)

-------------------------------------------------------------------------------------------------------------------

DAY 23, NOVEMBER 28: PURIFICATION OF NATIVE LEPA/EF 4 – GFP FUSION PROTEINS BY AFFINITY CHROMATOGRAPHY.

Purpose: In order to examine whether LepA/EF 4 – GFP fusion proteins retain function our prior experiments examined whether fusion proteins retained expression toxicity, and whether fusion proteins localized to the membrane. In order to examine other aspects of function, such as GTP – binding and ribosome - binding, purified protein is required. In this experiment we examine the feasibility of purifying LepA/EF 4 – GFP fusion proteins using affinity purification.

Background: Scientists are often interested in recovering proteins that have been produced in bacteria for research or therapeutic purposes. The process of protein purification from bacteria is medically relevant. It can be used as a method to mass-produce proteins of interest. For instance, the human insulin gene can be cloned into a plasmid and transformed into E. coli. The protein can then be purified and used to treat patients with diabetes.

The process of purifying a protein means separating this protein from other cellular components (proteins, DNA, ions, etc.). Protein purification is described in all biochemistry books, and in Chapter 5, Section A, of the textbook used in the Emmanuel College Biochemistry class (Fundamentals of Biochemistry, Voet, Voet and Pratt). This book is on reserve in the Emmanuel College Library. Here we will test the feasibility of using affinity chromatography, and magnetic beads to purify LepA/EF 4 fusion proteins in one step.

Preparation of magnetic beads for affinity purification of native LepA/EF 4. This procedure will be done the day before by the Emmanuel College Lab staff.

Background. The primary antibody that we employed to detect LepA/EF 4 – GFP in our Western blotting experiments can also be used to purify native, functional LepA/EF 4 – GFP fusion protein. Commercially available Protein A magnetic beads will be used for this purpose. Protein A is a very stable bacterial protein that has a high affinity for IgG produced from any animal species. Protein A magnetic beads contain tiny magnetized beads with Protein A covalently attached to their surface. Remember that the primary antibody that was employed in our prior experiments was affinity-purified rabbit IgG that was specific for GFP epitopes. The application of GFP primary antibody to Protein A magnetic beads will result in a tight, non-covalent attachment of GFP – specific IgG onto the magnetic beads. These beads would specifically recognize and bind to GFP that was present in a crude cell lysate. The application of a magnetic field could then be employed to collect the beads and the bound LepA/EF 4. By this method the collection and wash steps would be very rapid and very efficient. After washing away unbound proteins, purified LepA/EF 4 – GFP could be eluted from the magnetic beads. HOWEVER, the GFP IgG was non-covalently attached to the Protein A, and therefore some will leak out in the elution step. This would contaminate the purified LepA/EF 4 – GFP fusion protein with IgG. To avoid this problem, GFP IgG will be chemically cross-linked onto the Protein A magnetic beads by the procedure described in Sisson and Castor [Journal of Immunological Methods (1990), Vol. 127, 215-220].

1.

__________ are fairly small organelles that provide a safe place within the cell to carry out certain biochemical reactions that generate harmful, highly reactive oxygen species. These chemicals are both generated and broken down in the same location.

Lysosomes
Endosomes
Peroxisomes

Nucleosomes

2.

Biologists cannot possibly study all living species. Instead, they try to understand cell behavior by studying a select subset of them. Which of the following characteristics are useful in an organism chosen for use as a model in laboratory studies?

rapid rate of reproduction
all of the above
ability to grow under controlled conditions

amenability to genetic manipulation

3.

By definition, prokaryotic cells do not possess __________.

ribosomes.
a nucleus.
membrane bilayers.

replication machinery.

4.

Cell biologists employ targeted fluorescent dyes or modified fluorescent proteins in both standard fluorescence microscopy and confocal microscopy to observe specific details in the cell. Even though fluorescence permits better visualization, the resolving power is essentially the same as that of a standard light microscope because the resolving power of a microscope is limited by the __________ of light.

intensity
absorption
wavelength

filtering

5.

Choose the phrase that best completes this sentence: Microtubules ____________ and are required to pull duplicated chromosomes to opposite poles of dividing cells.

generate contractile forces
can rapidly reorganize
are found in especially large numbers in muscle cells

are intermediate in thickness

6.

Despite the differences between eukaryotic and prokaryotic cells, prokaryotes have proteins that are distantly related to eukaryotic actin filaments and microtubules. What is likely to be the most ancient function of the cytoskeleton?

cell division
cell motility
vesicle transport

membrane support

7.

DNA and RNA are different types of nucleic acid polymer. Which of the following is true of DNA but not true of RNA?

It contains thymine.
It has 5'-to-3' directionality.
It is single-stranded.

It contains uracil.

8.

Double covalent bonds are both shorter and stronger than single covalent bonds, but they also limit the geometry of the molecule because they ____________________.

prevent additional bonds from being formed with the bonded atoms.
limit the rotation of the bonded atoms.
create a new arrangement of electron shells.

change the reactivity of the bonded atoms.

9.

Drosophila melanogaster is a/an __________. This type of animal is the most abundant of all animal species, making it an appropriate choice as an experimental model.

mammal
amphibian
bird

insect

10.

If the isotope 32S has 16 protons and 16 neutrons, how many protons, neutrons, and electrons will the isotope 35S have, respectively?

16, 19, 17
16, 19, 15
16, 19, 16

16, 20, 15

11.

Living systems are incredibly diverse in size, shape, environment, and behavior. It is estimated that there are between 10 million and 100 million different species. Despite this wide variety of organisms, it remains difficult to define what it means to say something is alive. Which of the following can be described as the smallest living unit?

cell
organelle
protein

DNA

12.

Macromolecules in the cell can often interact transiently as a result of noncovalent interactions. These weak interactions also produce stable, highly specific interactions between molecules. Which of the factors below is the most significant in determining whether the interaction will be transient or stable?

the concentration of each molecule
the size of each molecule
surface complementarity between molecules

the rate of synthesis

13.

Many of the mechanisms that cells use for maintenance and reproduction were first studied at the molecular level in bacteria. Which bacterial species had a central role in advancing the field of molecular biology?

E. coli
S. pombe
C. elegans

D. melanogaster

14.

Many types of cells have stores of lipids in their cytoplasm, usually seen as fat droplets. What is the lipid most commonly found in these droplets?

cholesterol
isoprene
triacylglycerol

palmitic acid

15.

Mitochondria contain their own genome, are able to duplicate, and actually divide on a different time line from the rest of the cell. Nevertheless, mitochondria cannot function for long when isolated from the cell because they are __________________.

anaerobes.
viruses.
parasites.

endosymbionts.

16.

Mitochondria perform cellular respiration, a process that uses oxygen, generates carbon dioxide, and produces chemical energy for the cell. Which answer below indicates a correct pairing of material “burned” and the form of energy produced during cellular respiration?

fat, protein
sugar, fat
sugar, ATP
fat, ADP

17.

Polar covalent bonds are formed when the electrons in the bond are not shared equally between the two nuclei. Which one of these molecules contains polar bonds?

propane
methane
molecular oxygen
water

I JUST NEED SIMPLE/BASIC ANSWERS NOT LONG ANSWERS

1. Explain how life can be viewed at different levels of biological complexity.

2. Explain how researchers study biology at different levels, ranging from molecules to ecosystems.

3. Distinguish between discovery-based science and hypothesis testing, and describe the steps of the scientific method.

4. Tell whether a given topic or research study is about biology. Tell whether that study is discovery-based or hypothesis testing, or both.

5. If presented with a research study, propose what the Research Question was.

6. If presented with an example, tell whether a statement is a hypothesis, theory or law and why you think so.

Describe the general structure of atoms and their constituent particles.

Relate atomic structure to the periodic table of the elements. List the elements that make up most of the mass of all living organisms.

Explain how a single element may exist in more than one form, called isotopes, and how certain isotopes have importance in human medicine.

Compare and contrast the types of atomic interactions (bonds) that lead to the formation of molecules.

Tell which atoms and how many of each make up a given molecule (formula or diagram).

For a bond in a given molecule, tell whether the bond is covalent, and if it is, tell whether it is polar or nonpolar and why you think so.

Explain the concept of electronegativity and how it contributes to the formation of polar and nonpolar covalent bonds. Tell whether a given atom is electronegative or not, and why you think so. Label partial charges in a polar bond.

Draw five water molecules and label the atoms and bonds within and between the 5 molecules.

Describe how hydrogen bonding determines many properties of water.

List the properties of water that make it a valuable solvent, and distinguish between hydrophilic and hydrophobic substances.

Explain how water has the ability to ionize into hydroxide ions (OH–) and into hydrogen ions (H+), and how the H+ concentration is expressed as a solution's pH.

Explain the properties of carbon that make it the chemical basis of all life.

Describe the variety and chemical characteristics of common functional groups of organic compounds.

Diagram how small molecules may be assembled into larger ones by dehydration reactions and how hydrolysis reactions can reverse this process.

List the four major classes of organic molecules and macromolecules found in living organisms

If shown a molecule, tell whether it is organic or not and how you know.

If shown an organic molecule, tell whether it is an amino acid, nucleotide, fatty acid, steroid, monosaccharide or disaccharide and how you know.

Distinguish among different forms of carbohydrate molecules, including monosaccharides, disaccharides, and polysaccharides.

Relate the functions of plant and animal polysaccharides to their structure.

List the several different classes of lipid molecules important in living organisms.

Diagram the structure of a triglyceride and explain how it is affected by the presence of saturated and unsaturated fatty acids. Explain why some fats are solid at room temperature, and others are liquid.

Discuss how fats function as energy-storage molecules.

Apply knowledge of the structure of phospholipids to the formation of cellular membranes.

Describe the chemical nature of steroids and give an example of their biological importance.

Give examples of the general types of functions that are carried out in cells by different types of proteins.

Explain how amino acids are joined to form a polypeptide, and distinguish between a polypeptide and protein.

Describe the levels of protein structure and the factors that determine them. If shown a protein diagram, label parts that represent its levels of structure. Tell what kind of research techniques are used to determine protein structure.

Outline the bonding forces important in determining protein shape and function.

Explain what domains are and their importance in proteins.

Describe the three components of nucleotides.

Distinguish between the structures of DNA and RNA.

Tell how certain bases pair with others in DNA and RNA.

Discuss the 4 major categories of organic macromolecules found in living things, including structure, function and location of each.

Compare and contrast the general structural features of prokaryotic and eukaryotic cells.

Outline the differences in complexity among bacteria, animal, and plant cells.

Describe how a eukaryotic cell can be viewed as four interacting systems: the nucleus, cytosol, endomembrane system, and semiautonomous organelles.

Explain how the proteome underlies the structure and function of cells.

Analyze how cell size and shape affect the surface area/volume ratio.

Identify the location of the cytosol in a eukaryotic cell and list its general functions.

Describe the three types of protein filaments that make up the cytoskeleton. Identify which underlie cilia, flagella, centrioles, the nuclear lamina, and the brush border. Tell which are readily assembled and disassembled. Tell which are involved in various cell shape changes or movements.

Explain how motor proteins interact with microtubules or actin filaments to promote cellular movement.

Describe the structure and organization of the cell nucleus. Describe the structure and function of nuclear pores.

Outline the structures and general functions of the components of the endomembrane system.

Distinguish between the rough endoplasmic reticulum and the smooth endoplasmic reticulum.

Identify three important functions of the plasma membrane.

Tell why mitochondria and chloroplasts are called semi-autonomous organelles.

Discuss the evidence for the endosymbiosis theory.

Outline the structures and general functions of mitochondria and chloroplasts.

Explain how proteins are moved via vesicles through the endomembrane system.

If you are told about the function of a specific protein, tell where and in what type of cell that protein might be found.

Sketch and label a “typical” animal cell and a real animal cell such as a red blood cell or a muscle cell. Tell how it might be different than the “typical” cell in terms of its organelles and shape.

Sketch and label a “typical” plant cell and a real plant cell such as a root or leaf cell. Tell how it might be different than the “typical” cell in terms of its organelles and shape.

Describe the fluid-mosaic model of membrane structure. Tell why membranes are described as “fluid mosaics”.

Draw 10 phospholipid molecules as they would be arranged in a bilayer. Label the hydrophobic and hydrophilic parts.

61. Name all the places in a cell where membranes are found.

62. Describe at least three different categories of membrane proteins and tell what they do.

63. Describe what determines how fluid membranes are.

64. Explain how lipids are synthesized at the ER membrane.

65. Explain how transmembrane proteins are inserted into the ER membrane.

66. Compare and contrast diffusion, facilitated diffusion, passive transport, and active transport.

67. Explain the process of osmosis and how it affects cell structure.

68. Outline the functional differences between channels and transporters. Compare and contrast uniporters, symporters, and antiporters.

69. Explain the difference between primary active transport and secondary active transport.

70. Describe the structure and function of the sodium-potassium pump in animal cells and a similar pump in plants and prokaryotes.

71. Describe exocytosis and endocytosis and tell what molecules are moved across the membrane by these.

Discuss membrane transport, including its function and various forms and why all membranes have each form.

73. If a given cellular process is described, tell which form of transport might be involved.

List the different categories of animal tissue, providing general functions and specific examples of each.

Name the various organ systems found in many animals, list the components of each, and describe their general functions.

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