answer

watching

views

28 Feb 2018

It was discovered that a fraction of membrane proteins can be removed by immersing a membrane in an aqueous salt solution, whereas the remaining fraction of proteins can only be removed by immersing the membrane in a detergen solution or organic solvent which dissolves lipids.

Which fraction contains the peripheral proteins and which fraction contains the integral proteins and why? What does this experiment say about the way peripheral and integral proteins are bound to membranes?

answer

watching

views

For unlimited access to Homework Help, a Homework+ subscription is required.

You have 0 free answers left.
Get unlimited access to 3.8 million step-by-step answers.

Get unlimited access

Already have an account? Log in

Hubert KochLv2

2 Mar 2018

Unlock all answers

Get 1 free homework help answer.

Already have an account? Log in

Related questions

Question 401 pts

Kinetic energy is the stored energy that can be used for motion.

True

False

Flag this Question

Question 411 pts

When an electron is transferred from one atom to another, and the two atoms are then electrically attracted to one another, a(n) ________________ bond is formed.

Flag this Question

Question 421 pts

Changing the number of ____________ of an atom would change the chemical properties of the atom.

Flag this Question

Question 431 pts

Forming molecules and breaking down molecules in biological organisms usually requires the use of _________ to help the reaction proceed faster.

Flag this Question

Question 441 pts

Organic molecules have a core composed of .

Flag this Question

Question 451 pts

The building blocks of carbohydrates are

Flag this Question

Question 461 pts

Water molecules crossing a membrane from high to low concentration is .

active transport

cell fate

facilitated diffusion

osmosis

Flag this Question

Question 471 pts

Choose the membrane molecule responsible for the passage of polar molecules and ions into and out of the cell.

phospholipids

transmembrane channel proteins

cell surface proteins

carbohydrate chains

Flag this Question

Question 481 pts

The simplest cells are .

Flag this Question

Question 491 pts

Which of the following membrane bound organelles are found inside bacterial cells:

nucleus

organelles are not found in prokaryotic cells

mitochondria

chloroplasts

Flag this Question

Question 501 pts

Which of the following is a specialized components of the cell, is associated with the rough endoplasmic reticulum, and is responsible for making proteins.

Flag this Question

Question 511 pts

Proteins are sorted, modified, and packaged by the _____________ and later transported to the outside of the cell.

ribosomes

golgi bodies (golgi apparatus)

nucleus

mitochondria

Flag this Question

Question 521 pts

Dehydration synthesis is a process of linking two smaller subunits together to form a polymer. Which of the following statements below is true of dehydration synthesis?

Oxygen is consumed.

A water molecule is removed from the molecules.

A water molecule is added to the molecules

Carbon dioxide is given off.

Flag this Question

Question 531 pts

Proteins are made up of _________ held together by peptide bonds.

Flag this Question

Question 541 pts

Which of the following gives an amino acid its properties?

NH2

R Group or functional group

COOH

Flag this Question

Question 551 pts

The final three-dimensional shape of a protein that includes the bonding of two or more polypeptide chains is call its _____ structure.

Flag this Question

Question 561 pts

How do DNA and RNA differ?

all statements are differences between DNA and RNA.

DNA is double stranded while RNA is single stranded.

They have different sugars.

Thymine is present in DNA but not in RNA.

Flag this Question

Question 571 pts

Which of the following is a component of a DNA nucleotide?

phosphate

a nitrogen containing base

All are components of a nucleotide.

5-carbon sugar

Flag this Question

Question 581 pts

Cell membranes are made up of several different types of molecules. Select the membrane molecule below that is made up of a polar region and two non-polar fatty acid tails.

transmembrane proteins

carbohydrate chains

phospholipids

cell surface proteins

Flag this Question

Question 591 pts

Two organelles which are believed to have once been free-living bacterial cells are ____________ and ______________.

chloroplasts and mitochondria

golgi apparatus and endoplasmic reticulum

ribosomes and nucleolus

peroxisomes and lysosomes

Flag this Question

Question 601 pts

The is an extensive system of internal membranes responsible for producing carbohydrates and lipids.

smooth endoplasmic reticulum

rough endoplasmic reticulum

nucleolus

mitochondria

sangriarat617

Biochemistry Protocols: Your First Protein Purification

As the newest student in a biochemistry research lab, you are given responsibility for purifying a mitochondria protein. Following a protocol for the purification, you proceed through the steps below. As you work, a more experienced student questions you about the rationale for each procedure. Supply the answers.

(a) You pick up 20 kg of beef hearts from a nearby slaughterhouse. You transport the hearts on ice, and perform each step of the purification on ice or in a walk-in cold room. You homogenize the beef heart tissue in a high-speed blender in a medium containing 0.2 M sucrose, buffered to a pH of 7.2.

Why do you use beef heart tissue, and in such large quantity?

What is the purpose of keeping the tissue cold and suspending it in 0.2 M sucrose, at pH 7.2? What happens to the tissue when it is homogenized?

(b) You subject the resulting heart homogenate, which is dense and opaque, to a series of differential centrifugation steps. What does this accomplish?

(c) You proceed with the purification using the supernatant fraction that contains mostly intact mitochondria. Next you osmotically lyse the mitochondria. The lysate, which is less dense than the homogenate, but still opaque, consists primarily of mitochondrial membranes and internal mitochondrial contents. To this lysate you add ammonium sulfate, You centrifuge the solution, decant the supernatant, and discard the

pellet. To the supernatant, which is clearer than the lysate, you add more ammonium sulfate. Once again, you centrifuge the sample, but this time you save the pellet because it contains

the protein of interest. What is the rationale for the two-step addition of the salt?

(d) You solubilize the ammonium sulfate pellet containing the mitochondrial proteins and dialyze it overnight against large volumes of buffered (pH 7.2) solution. Why isnât ammonium sulfate included in the dialysis buffer? Why do

you use the buffer solution instead of water?

(e) You run the dialyzed solution over a size-exclusion chromatographic column. Following the protocol, you collect the first protein fraction that exits the column, and discard the rest of the fractions that elute from the column later. You detect the protein by measuring UV absorbance (at 280 nm) in the fractions. What does the instruction to collect the first fraction tell you about the protein? Why is UV absorbance at 280 nm a good way to monitor for the presence

of protein in the eluted fractions?

(f) You place the fraction collected in (e) on a cation exchange chromatographic column. After discarding the initial solution that exits the column, you add a washing solution of higher pH to the column and collect the protein fraction that immediately elutes. Explain what you are doing.

(g) You run a small sample of your fraction, now very reduced in volume and quite clear (though tinged pink), on an isoelectric focusing gel. When stained, the gel shows three sharp bands. According to the protocol, the protein of interest is the one with the pI of 5.6, but you decide to do one more assay of the proteinâs purity. You cut out the pI 5.6 band

and subject it to SDS polyacrylamide gel electrophoresis. The protein resolves as a single band. Why were you unconvinced of the purity of the âsingleâ protein band on your

isoelectric focusing gel? What did the results of the SDS gel tell you? Why is it important to do the SDS gel electrophoresis after the isoelectric focusing.

purplemouse591

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; âprotein purification.pptâ

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

5. What is âProtein Aâ and how did we use it?

6. What antibody did we use in our purification experiment and why did we use it? Why canât 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 30Â°C VS. 37Â°C

Purpose: Compare the growth of pPEM109, pIN III, and candidate plasmids in DH5alpha-FâlacI^Q -/+ 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, lacI^Q, 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 lacI^Q gene contains a âpromoter-upâ mutation that increases transcription and Lac repressor expression 10-fold, resulting in ~100 repressor protein molecules per wild-type cell (MÃ¼ller-Hill et al., 1998). Strains that only have normal Lac repressor have a slightly elevated level of âleakyâ expression from the lac promoter. It was shown by comparing two identical strains (one contained lacI^Q and one contained lacI) that pPEM109 could only be maintained in the lacI^Q 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?

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

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?

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

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

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

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 â GFP FUSION PROTEINS BY AFFINITY CHROMATOGRAPHY.

Purpose: In order to examine whether LepA/EF 4 â 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 â binding and ribosome - binding, purified protein is required. In this experiment we examine the feasibility of purifying LepA/EF 4 â 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 â GFP in our Western blotting experiments can also be used to purify native, functional LepA/EF 4 â 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 â 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 â 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 â 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].