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Molecular Biology > 4-Protein Detection & Analysis > Flashcards

4-Protein Detection & Analysis Flashcards

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1
Q

Which are the 3 techniques that allow us to examine proteins?

A
  1. Chromatography
  2. Proteomics
  3. Immuno-detection( Western blotting & Immunofluorescence)
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2
Q

Why we Analyse Proteins?

A
  • Proteins play a crucial role in nearly all biological processes, so its important to understand how the function
  • To explore how amino acids sequencies specify the proteins structure and how these proteins are bind together in order to perform their function.
  • Pharmaceutical companies design activators or inhibitors.
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3
Q

Which are the main steps in protein analysis?

A

1) Extraction of proteins
2) Purification of the protein
3) Structural characterisation of the protein

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4
Q

What protein extraction involves?

A
  • Involves the extraction of the protein or the peptide in a biological active form from tissue
  • During this process deactivation of proteolytic enzymes is required , in order not to cause degradation of the protein contained in the tissue.
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5
Q

How can we extract a protein?

A

homogenize it in a physiological buffer (0.05M NaPO4, PH 7.4 containing proteolytic enzyme inhibitors (EDTA, Pepstatin)

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6
Q

How can we extract peptides?

A

boil the tissue with 1M Acetic Acid for 5 min homogenize tissue in ethanol / 0.1MHCl (ratio 3:1) at 0°C. This will burst open the vesicles to release the peptides in solution.

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7
Q

How can proteins be purified? (with what criteria)?

A

Proteins can be purified depending on different properties they process. These properties allow us to develop new techniques in purifying proteins.

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8
Q

What technique we use if we want to purify proteins depending on Solubility?

A

Differential Precipitation

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9
Q

What technique we use if we want to purify proteins depending on Molecular Size?

A

Gel Filtration Chromatography

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10
Q

What technique we use if we want to purify proteins depending on Molecular Charge?

A

Ion Exchange Chromatography

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11
Q

What technique we use if we want to purify proteins depending on Hydrophoicity?

A

Reversed Phase HPLC

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12
Q

What technique we use if we want to purify proteins depending on Biological Activity?

A

Affinity Chromatography

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13
Q

What does precipitaion means?

A

The process of formation of a solid that was previously held in a solution (Καθίζηση).

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14
Q

What is the function of NH4SO4 and Polyethylene glycol?

What if the concentrations of these substancs are increased?

A

Substances that If applied into a solution with proteins, cause propitiation which can be separated with centrifugation.

If the concentrations of these two substances are increased, even more precipitate forms.

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15
Q

Describe Gel Filtration Chromatography (INFO)

A

 Also called Gel Permeation Chromatography
 Seperates protein molecules according to their molecular size
 The solution is inserted to the top of a specialized column.
 This column consists of specialized porous heads
 Small molecules of protein enter the beads while large molecules can’t stay and stay in the space between the beads
 Therefore, large molecules flow more rapidly through the column and emerge first from the bottom of the column

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16
Q

What the advantage and dissadvantage of Gel Filtration Chromatography?

A

 ADVANTAGE: Larger quantities of proteins can be separated

 DISADVANTAGE: Lower resolution

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17
Q

Describe Ion Exchange Chromatography (INFO)

A

 Separates protein molecules according to their molecular charge.
 In this technique, the beads of the column have a specific charge on them. This is a result of a molecule that is attached to these beads

 +ve charged beads: Beads that are attached to DEAE ( Diethylaminoehtyl) cellulose

 -ve charged beads: Beads that are attached to carboxymethyl cellulose

 The beads on the column depend on the protein we want to purify

 In the protein is -ve charged we have to use positive charged beads and vice versa.

How does it work?
 For example, if the protein of interest is negatively charged, then we will use DEAE- cellulose column.
 The protein will bind to the positively charged beads
 This protein that is attached to the beads can be released by increasing the concentration of NaCl( or another salt).
 The Na+ ions (or other cation) will compete to bind to the beads on the column instead of the protein.
 Proteins that are highly positively charged will emerge first because they will be repelled by the beads.

18
Q

Describe High Performance Liquid Chromatography (INFO)

A

• Separation is based on the analyte’s( chemical of interest) relative solubility between two liquid phases

HPLC consists of 2 modes:
1. Normal Phase:
 Polar stationary phase
 non-polar solvent

  1. Reverse Phase (more in use):
     Non-polar stationary phase
     a polar solvent.

Before HPLC:
• The peptide is first hydrolysed into its constituent amino acids by heating it in 6M HCI at 110 °C for 24 hours.
• The amino acids are then separated by HPLC
• The react with ninhydrin, a compound that makes amino acids change colour.

19
Q

Describe Affinity Chromatography(AC) (INFO)

A

 AC involves an inert matrix coupled with a affinity ligand specific for a binding site on the target molecule. (Like antisomes with antigens)
 Under specific conditions this affinity matrix will bind molecules according to its specificity only.
 All other components will pass through the column unabsorbed.
 After washing (the unabsorbed molecules have exited the column), conditions (ph , temperature etc) are changed in order to disassociate or displace the molecules from the ligand
 Simple ‘’on-off’ mode is applied by switching abruptly from full binding to complete release conditions.

20
Q

Which and how many steps are ivolved in Protein Characterization ?

A
  1. Determining the amino acid composition(finding out the amino acids that the protein is made of and their number)
  2. Determining the Amino Acid Sequence( involves finding out the sequence of amino acids of proteins in order.
  3. Determining the Molecular Mass of the Protein
21
Q

Describe Edman’s degradation method

A

Lets say that we have a protein containing 3 amino acids. In this method, we try to break down one amino acid pair time with a specific procedure in order to examine it and define which amino acid it is. When we cut off the first amino acid, the protein remains with 2 amino acids

Its more effective than the Sanger method, because the bindings (glycosidic bonds) between amino acids are not affected, whereas in the Sanger method we hydrolyse the protein and as a result we can isolate the first amino acid but in the protein the remaining peptide bonds are destroyed and the amino acids are scrambled. ( we cannot define which amino acid is the 1st, 2nd, 3rd, etc.)

 Longer polypeptide chains are broken into shorter ones for analysis by specifically cleaving them with enzymes that cleave at specific points

22
Q

Describe X-ray crystallography

A

 X-ray crystallography is a method used for determining the atomic and molecular structure of a crystal, in which the crystalline atoms cause a beam of X-rays to diffract into many specific directions.

 Measuring the angles and intensities of these beams, a crystallographer can produce a 3-D picture of the density of electrons within the crystal.

 From this electron density, the chemical bonds, the mean positions in the crystal can be determined, their disorder and various other information.

23
Q

Describe Nuclear Magnetic Resonance Spectroscopy (NMR)

A
  • X-ray crystallography requires crystals and many proteins are difficult for this.
  • NMR spectroscopy, overcomes this problem and exploits the magnetic properties of certain atomic nuclei.
  • NMR spectroscopy is used to investigate the properties of complex proteins
  • This kind of procedure is applicable in any sample that contains spinning nuclei, not just proteins.
  • NMR spectroscopy is crucial because it can be applied to a diversity of samples ( solids, solutions) and gives us a range of information.
24
Q

What is Spectrometry?

A

The procedure of observing and measuring the wavelengths of light or other electromagnetic emissions

25
Q

What Mass Spectrometry involves and what does it depend on ?

A

involves an ionization source, a mass analyser and a detector.

It depends on one principle: The smaller ions will fly faster and hit the detector first, then the larger ions reach. The ions hit the detector in order by mass

.Flight time of the ions make it possible to determine molecular masses.

26
Q

What is Matrix-assisted laser desorption/ionization (MALDI) Describe it (INFO).

A

Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules.

 Method where a laser is used to generate ions of high molecular weight samples, such as proteins and polymers.
 Analyte is embedded in to crystal matrix
 The presence of an aromatic matrix causes the large molecules to ionize instead of decomposing.

27
Q

What is a proteome?

A

• The proteome is the complete complement of proteins found in a complete genome or specific tissue.

28
Q

Describe Gel Electrophoresis (INFO)

A

• Molecules are divided based on their size
• Gel Electrophoresis is used to divide molecules.
 Big (in size) molecules travel slower.
 Small (in size) molecules travel faster
• Polyacrylamide gel provides a porous matrix (PAGE)—Polyacrylamide Gel Electrophoresis
• Sample is stained with comassie blue to make it visible in the gel

29
Q

Describe 2-D Gel Electrophoresis (INFO)

A
  • 2-D Separation is based on size and charged
  • First step is to separate based on charge or isoelectric point, called isoelectric focusing.
  • Then separate based on size Soldium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE)
30
Q

Describe 2-D Proteomics Analysis

A

• Proteins of interest can also be excised from the gel and sent on to mass spectrometry for definitive identification.

31
Q

What is Protein Sequencing?Which are the two major methods?

A
  • Protein Sequencing is a technique to determine the amino acid sequence of a protein
  • Two major direct methods of protein sequencing are mass spectrometry and the Edman degradation reaction.
  • Edman degradation protein sequencing allows the ordered amino acid composition of a protein to be discovered.
32
Q

What an antibody is composed of?

A

Antibody Structure:

1) 2 heavy chains + 2 light chains
2) Disulphide bonds
3) 2 antigen binding sites

33
Q

Name some antibody isotypes.

A

i. IgG
ii. IgM
iii. IgA
iv. IgE
v. IgD

34
Q

How B cell clones are activated?

A

They are activated by a specific target antigen

35
Q

What B cell clones do when they connect with the target antigen?

A

They secrete specific antibodies

36
Q

How can we produce antibosies using an animal? (INFO)

A
  • Inject antigen (i.e purified protein) into an animal (mouse, chicken etc)
  • Animal produces antibodies that recognize antigen

Antigen injected more than once: response heightened in subsequent injections

37
Q

Where polyclonal antibodies are derive from and how they function against an antigen?

A

Derived from multiple B-cell clones

-recognize multiple epitopes on antigens.

38
Q

Where monoclonal antibodies are derive from and how it functions against an antigen?

A

Derived from B-cell clone ‘’Hybridoma’’.

Recognises single epitope on antigen.

39
Q

Uses of antibodies in molecular biology (INFO)

A 
Western Blotting (Immunoblotting):
Identification of protein antigen following SDS-PAGE

Immunoprecipitation:
Isolation of specific proteins + binding partners

Immunofluorescence microscopy:
Localisation of specific proteins in cells

ELISA( Enzyme-Linked Immunosorbent Assay):
Detection of proteins in a sample

40
Q

Which are the steps for Immunoprecipitation

Identification of protein-protein interactions? (INFO)

A
  1. Attach antibody to beads via protein A
  2. Lyse cells to release antigen and its binding parts
  3. Mix cell lysate + antibody-coated beads (antibody binds antigen)
  4. Purify antigen and its binding partners by centrifugation
41
Q

Why ELISA method is used?

A

ELISA method is used for detection of proteins (i.e cytokines, HIV antigens) in samples

Molecular Biology (9 decks)

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