Ch 5 Techniques in Protein Biochemistry

PPT 10 Can proteins be purified according to their solubility?

yes

PPT 11 Can proteins be purified according to their size?

yes

PPT 18 Can proteins be separated by gel electrophoresis and displayed?

yes

PPT 25 Can a purification scheme be quantitatively evaluated?

yes

PPT 17 Graph of Gel Filtration by HPLC

). Gel filtration by HPLC clearly defines the individual proteins because of its greater resolving power. Proteins are detected by their absorbance of 220-nm light waves: (1) thyroglobulin (669 kDa), (2) catalase (232 kDa), (3) bovine serum albumin (67 kDa), (4) ovalbumin (43 kDa), and (5) ribonuclease (13.4 kDa). [After K. J. Wilson and T. D. Schlabach. In Current Protocols in Molecular Biology, vol. 2, suppl. 41, F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, Eds. (Wiley, 1998), p. 10.14.1.]

PPT 4 Lactate dehydrogenase - what is it and why do we use it?

- An assay for the enzyme lactate dehydrogenase is based on the that a product of the reaction, NADH, can be detected spectrophotometrically .... Proteins can be purified on the basis of difference in their chemical properties

PPT 40 Describe the process of how the estrogen receptor can be purified by immonoprecipitation?

- a monoclonal antibody for the estrogen receptor can be isolated by searching for cell lines that produce an antibody that binds to the receptor - In an antibody for the receptor is present, it will bind to the receptor and alter the sedimentation constant of the receptor

PPT 7 How do we remove protein from cells to be purified?

- cells are disrupted to for a hemogenate, which is a mixture of all of the components of the cell but no intact cells - The homogenate is then centrifuged at low speed to yield a pellet consisting of nuclei and a supernatant. This supernatant is then centrifuged at a higher centrifugal force to yield another pellet and supernatant. -This process, called differential centrifugation, is repeated several more times to yield a series of pellets enriched in various cellular material and a final supernatant called the cytosol

What physical difference among proteins allow for their purification ?

- charge - size - binding affinity - solubility?

PPT 20 Proteins can be separated by gel electrophoresis and displayed

- proteins separated by SDS-PAGE are visualized by staining the gel with dyes such as coomassie blue

PPT 22 What procedure is the following describing? -In _______________________________, proteins are separated in one direction by isoelectric focusing. This gel is then attached to an SDS-PAGE gel, and electrophoresis is performed at a 90° angle to the direction of the isoelectric focusing separation.

-In two-dimensional gel electrophoresis, proteins are separated in one direction by isoelectric focusing. This gel is then attached to an SDS-PAGE gel, and electrophoresis is performed at a 90° angle to the direction of the isoelectric focusing separation.

ion exchange chromatography

-stationary phase is made of either negatively or positively charged beads (attract & bind compounds that have opposite charge) -salt is added to elute proteins stuck to column

PPT 2 Outline

5.1 - The proteome is the functional representation of the genome 5.2 - The purification of protein is the first step in understanding their function 5.3 - Immunological techniques are used to purify and characterize proteins 5.4 - Determination of primary structure facilitates an understanding of protein function

PPT 11 Molecular exclusion chromatography (gel filtration chromatography) allows the separation of proetins on the basis of size... describe the process

A column is filled with porous beads. When a protein solution is passed over the beads, large protein cannot enter the beads and exit the column first. Small proteins can enter the beads and thus have a longer path and exit the column last

PPT 12 Model of Gel-filtration chromatography

A mixture of proteins in a small volume is applied to a column filled with porous beads. Because large proteins cannot enter the internal volume of the beads, they emerge sooner than do small ones

Antigen

A protein that, when introduced in the blood, triggers the production of an antibody

antigenic determinants

A region on the surface of an antigen molecule to which an antibody binds.

isoelectric focusing

A specialized method of separating proteins by their isoelectric point using electrophoresis; the gel is modified to possess a pH gradient

PPT 23 Diagram of two-dimensional gel electrophoresis

A) A protein sample is initially fractionated in one direction by isoelectric focusing as described in Figure 5.10. The isoelectric-focusing gel is then attached to an SDS-polyacrylamide gel, and electrophoresis is performed in the second direction, perpendicular to the original separation. Proteins with the same pI value are now separated on the basis of mass.

PPT 19 Diagram of Polyacrylamide Gel Electrophoresis (SDS-PAGE)

A) Gel-electrophoresis apparatus. Typically, several samples undergo electrophoresis on one flat polyacrylamide gel. A microliter pipette is used to place solutions of proteins in the wells of the slab. A cover is then placed over the gel chamber, and voltage is applied. The negatively charged SDS (sodium dodecyl sulfate)-protein complexes migrate in the direction of the anode, at the bottom of the gel. (B) The sieving action of a porous polyacrylamide gel separates proteins according to size, with the smallest moving most rapidly.

PPT 14 Can proteins be purified according to binding affinity

Absolutely

PPT 14 Affinity chromatography explained

Affinity chromatography takes advantage of the fact that some proteins have a high affinity for specific chemicals or chemical groups. Beads are made with the specific chemical attached. A protein mixture is passed through the column. Only protein with affinity for the attached group will be retained. The bound protein is then released by passing a solution enriched in the chemical to which the protein is bounds.

PPT 35 What is an antibody?

An antibody is a protein synthesized in response to the presence of a foreign substance called an antigen

PPT 37 antibodies that recognize only one epitope

An antibody-producing cell synthesizes antibodies that recognize only one epitope. Each antibody-producing cell thus synthesizes a monoclonal antibody

Why is an assay required for protein purification?

An assay, which should be based on some unique biochemical property of the protein that is being purified, allows the detection of the protein of interest.

gradient centrifugation

An ultracentrifugation technique used to separate large biomolecules or molecular complexes. A linear gradient of viscous solution is created in a centrifuge tube—for instance, a gradient of 5% to 20% sucrose. The sample is layered on the top of the gradient, and the tube is centrifuged at high speeds. Large, dense molecules or complexes move through the gradient faster than smaller complexes. The separated complexes are harvested by making a hole in the bottom of the tube and collecting drops. Also called zonal centrifugation.

PPT 37 antibodies produced to the antigen by different cells

Any antigen may have multiple epitopes.The antibodies produced to the antigen by different cells are said to be polyclonal

PPT 38 Did you know?

Clinical laboratories use monoclonal antibodies in many assays. For example, the detection in the blood of enzymes that are normally localized in the heart points to a myocardial infarction (heart attack). Blood transfusions have been made safe by antibody screening of donor blood for viruses that causes AIDS, hepatitis, and other infectious diseases. Monoclonal antibodies also find uses as therapeutic agents. Trastuzumab (Herceptin), for example, is a monoclonal antibody useful for treating some forms of breast cancedr

Preteome

Complete protein composition of a cell the entire set of proteins expressed by a given cell, tissue, or organism The full range of proteins a cell is able to produce.

PPT 10 Which procedure is being described......"The protein solution is placed in a cellophane bag with pores too small to allow the protein to diffuse but big enough to allow the salt to equilibrate with the solution surrounding the __________ bag"

DThe salt can be removed from a protein solutions by dialysis

PPT 8 How are proteins removed from the cells? (term)

Differential centrifugation

PPT 24 Images of alteration in protein levels detected by two-dimensional gel electrophoresis

Figure 5.12 Alterations in protein levels detected by two-dimensional gel electrophoresis. Samples of (A) normal colon mucosa and (B) colorectal tumor tissue from the same person were analyzed by two-dimensional gel electrophoresis. In the gel section shown, changes in the intensity of several spots are evident, including a dramatic increase in levels of the protein indicated by the arrow, corresponding to the enzyme glyceraldehyde-3-phosphate dehydrogenase. [Courtesy of Lin Quinsong © 2010, The American Society for Biochemistry and Molecular Biology.]

PPT 27 Diagram of electrophoretic analysis of a protein purification

Figure 5.13 Electrophoretic analysis of a protein purification. The purification scheme in Table 5.1 was analyzed by SDS-PAGE. Each lane contained 50 μg of sample. The effectiveness of the purification can be seen as the band for the protein of interest becomes more prominent relative to other bands.

PPT 39 Diagram or the preparation of monoclonal antibodies

Figure 5.19 The preparation of monoclonal antibodies. Hybridoma cells are formed by the fusion of antibody-producing cells and myeloma cells. The hybrid cells are allowed to proliferate by growing them in selective medium. They are then screened to determine which ones produce antibody of the desired specificity. [After C. Milstein. Monoclonal antibodies. Copyright © 1980 by Scientific American, Inc. All rights reserved.]

PPT 32 Gradient centrifugation provides and _________ for the ________-___________ complex

Gradient centrifugation provides an assay for the Estradiol-Receptor complex

PPT 21 How does isoelectric focusing work?

IT allows separation of proteins in a gel on the basis of their relative amounts of acidic or basic amino acids. If a mixture or proteins is placed in a gel with a pH gradient and an electrical field is applied, proteins will migrate until they reach their isoelectric point, the pH which they have no net charge

PPT 4 What is lactate dehydrogenase ?

Lactate dehydrogenase is an enzyme found in nearly all living cells. LDH catalyzes the conversion of lactate to pyruvate and back, as it converts NAD⁺ to NADH and back. A dehydrogenase is an enzyme that transfers a hydride from one molecule to another. LDH exists in four distinct enzyme classes. The lactate dehydrogenase (LDH) test looks for signs of damage to the body's tissues. LDH is an enzyme found in almost every cell of your body, including your blood, muscles, brain, kidneys, and pancreas. The enzyme turns sugar into energy. The LDH test measures the amount of LDH in your blood or other body fluid

PPT 11 What is the procedure called that allow protein to be purified by their size?

Molecular exclusion chromatography

differential centrifugation

Procedure for separating cellular components according to their size and density by spinning a cell homogenate in a series of centrifuge runs. After each run, the supernatant is removed from the deposited material (pellet) and spun again at progressively higher speeds. ** Cells disrupted in a homogenizer, and the resulting mixture, called the homogenate, is centrifuged in a step-by-step fashion of increasing centrifugal force. The denser material will form a pellet at lower centrifugal force than will the less dense material. The isolated fraction can be used for further purification

gel electrophoresis

Procedure used to separate and analyze DNA fragments by placing a mixture of DNA fragments at one end of a porous gel and applying an electrical voltage to the gel

salting out

Protein precipitation caused by an increase in the salt concentration.

PPT 4 Does protein purification require a test?

Protein purification requires a test, or assay, that determined whether the protein of interest is present

PPT 21 Isoelectric focusing

Proteins can separated by gel electrophoresis and displayed

PPT 9 Salting in and salting out explained

Salting out takes advantage of the fact that the solubility of proteins varies with the salt concentration. Most proteins require some salt to dissolve in water, a process called salting in. As salt concentration is increased, different proteins will precipitate at different salt concentrations, a process called salting out

PPT 35 How does an antibody bind to a protein?

The antibody recognizes a particular structural feature on the antigen called the antigenic determinant or epitope

PPT 25 How can a purification scheme be quantitatively evaluated and what allows for visualization?

The effectiveness of a purification scheme is measure by calculating the specific activity after each separation technique SDS-PAGE allows a visual evaluation of the purification scheme

What are the different methods used to purify proteins?

The four methods of protein purification are: (1) Extraction (2) Precipitation and Differential Solubilisation (3) Ultracentrifugation and (4)Chromatographic Methods. The methods used in protein purification, can roughly be divided into analytical and preparative methods.

PPT 3 The ___________ is The entire set of proteins is expressed and modified by a call under a particular set of biochemical condition

The precome is the entire set of protein expressed and modified by a call under a particular set of biochemical conditions

PPT 4 Section 5.2 What is the first step in understanding the function of proteins?

The purification of proteins is the first step in understanding their function

salting in

The solubility of a protein at low ion concentrations increases as salt is added.

PPT 4 What is learning objective 4: ?

To explain how proteins can be purified

monoclonal cell line

What is monoclonality? Monoclonality is term that describes a cell line that originates from a single progenitor (single cell) - and is therefore monoclonal. Cell line development and assurance of monoclonality are critical steps in the process of generating biopharmaceutical molecules, such as monoclonal antibodies.

PPT 35 Can antibodies to specific proteins be generated?

Yes

PPT 40 Can the estrogen receptor be purified by immonsupression?

Yes

PPT 42 Can antibodies be used to purify the estrogen receptor?

Yes The estrogen receptor can be purified by immunoprecipitation - Once the monoclonal cell line is isolated, the antibody can be used to purify the estrogen receptor

monoclonal antibodies

a collection of identical antibodies that interact with a single antigen site

two-dimensional gel electrophoresis

a laboratory method that separates proteins according to their isoelectric points and molecular weights

dialysis

a procedure to remove waste products from the blood of patients whose kidneys no longer function

polyclonal antibodies

a series of antibodies are produced responding to a variety of different sites on the antigen

What is a hemogenate?

a suspension of cell fragments and cell constituents obtained when tissue is homogenized.

PPT 18 What does Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) allow?

allows accurate determination of mass. SDS denatures proteins, and for most proteins, 1 molecule of SDS binds for every two amino acids. Thus, proteins have the same charge-to-mass ratio and migrate in the gel on the basis of mass only.

PPT 7 In order to be purified proteins must....

be removed from the cell to be purified

PPT 31 Immunological techniques are used to purify and

characterize proteings

PPT 13 Ion exchange chromatography allows separation of proteins on the bases of ____________

charge

PPT 13 The beads in ion-exchange chromatography are

charged

lactate dehydrogenase

converts pyruvate to lactate

SDS-PAGE

denatures the proteins and masks the native charge so that comparison of size is more accurate, but the functional protein cannot be recaptured from the gel

PPT 5 Protein purification are monitored in part by....

determining the specific activity of the protein being used

PPT 10 The salt can be removed from a protein solution by

dialysis

PPT 29 Ultracentrifugation can be used to ...

examine proteins

PPT 11 What is another name for molecular exclusion chromatography?

gel filtration

PPT 13 In ion exchange chromatography.... proteins with the opposite charge will bind to the beads and are subsequently released by

increasing the salt concentration or adjusting the pH of the buffer that is passed through the column

PPT 13 Proteins can be purified according to their charge using...

ion-exchange chromaography

PPT 13 In ion exchange chromatography....when a mixture of proteins is passed through the column...

proteins with the same charge as on the column will exit the column quickly

PPT 16 How would we increase the number of interactions when purifying according to solubility, size, charge, and binding affinity

put those bad boys under high pressure to increase the amount of interactions... explained below •Proteins can be purified according to solubility, size, charge, and binding affinity. -The resolving power of any chromatographic technique is related to the number of potential sites of interaction between the protein and the column beads. Very fine beads allow more interactions and thus greater resolving power, but flow rates through such columns are too slow. -High-pressure liquid chromatography (HPLC) uses very fine beads in metal columns and high-pressure pumps to move the liquid through the column. Because of the increased number of interaction sites, the resolving power of HPLC is greater than normal columns.

PPT 5 In the case of an enzyme purification, specific activity is the ....

ration of enzyme activity to protein concentration...

PPT 29 Sedimentation coefficient, S formula

s = m (1 - v̅ ρ) /ƒ Where m = mass, v̅ = the partial specific volume (the reciprocal of the particle density), ρ = density of the medium, and ƒ = the frictional coefficient of the particle Sedimentation coefficients are usually expressed as Svedberg units (S) equal to 10−13 s. The smaller the S value, the slower the protein moves in a centrifugal field.

A monoclonal cell line is isolated by

screening for the antibody of interest

PPT 41 Graph of the alteration of the sedimentation profile of an antibody

sedimentation profile when antibody binds to the receptor protein. The estradiol-receptor-antibody complex migrates farther into the centrifuge tube because it is larger than the estradiol-receptor complex alone. Abbreviations: DPM, disintegrations per minute; ER, estradiol-receptor complex; mab, monoclonal antibody.

PPT 29 Centrifugation is a means of

separating proteins

PPT 29 in ultracentrifugation, the smaller the S values, the ______________ the proteins moves in a centrifugal field

slower

PPT 9 Proteins can be purified according to

solubility, size, charge, and binding affinity

PPT 5 Specific activity should________________ with each step of the purification procedure

specific activity should increase with each step of purification procedure

high pressure liquid chromatography

the column and solution use an apparatus that puts the system under high pressure

PPT 29 Ultracentrifugation can be used to examine proteins....when subjected to a centrifugal force....

the rate of movement of the particle is defined by the sedimentation coefficient,S

PPT 20 Why would we stain gels with Dyes?

to visualize the protein

PPT 3 Unlike the genome, the pretome is ________ an unvarying characteristic of the cell

unlike the genome, the pretome is not an unvarying characteristic of the calls

affinity chromatography

uses a bound receptor or ligand and an eluent with free ligand or a receptor for the protein of interest

PPT 32 Entire PPT

•A density gradient is formed in a centrifuge tube, and a mixture of proteins in solution is placed on top of the gradient. •To identify the estradiol receptor, the protein mixture is first incubated with radioactive estradiol, which is readily detected. Only the estradiol receptor will bind to the steroid. Moreover, the steroid alone is too small to be influenced by the centrifugal force. •After the centrifugation is complete, a small hole is made in the bottom of the centrifuge tube and portions of the gradient are collected and tested for radioactivity.

PPT 38 Monoclonal antibodies with virtually any desired specificity can be readily prepared

•Immortal cell lines producing monoclonal antibodies can be generated by fusing normal antibody- producing cells with cells from a type of cancer called multiple myeloma. •A monoclonal cell line is isolated by screening for the antibody of interest.

PPT 18 Proteins will migrate in an electrical field because they are _________. When the migration occurs in a gel, the process is called ___________ ______________

•Proteins will migrate in an electrical field because they are charged. When the migration occurs in a gel, the process is called gel electrophoresis.

PPT 31 Learning objective 5: Explain how immunological techniques can be used to purify and identify proteins

•The estrogen receptor binds the steroid hormone estradiol tightly and with great specificity. •The estrogen receptor has no enzymatic activity but can be purified by immunological techniques and the use of gradient centrifugation.