Unit 1- Part 1 Flashcards
(148 cards)
1
Q
What should happen before any activity is carried out in the Laboratory?
A
The protocol should first be considered in terms of hazards and risk.
2
Q
What is a hazard?
A
Anything that poses a potential threat to an individual or the environment.
3
Q
Describe different types of hazards in the Laboratory.
A
Toxic or corrosive chemicals, heat or flammable substances, pathogenic organisms and mechanical equipment.
4
Q
What is a risk?
A
Risk is the likelihood of harm arising from exposure to a hazard.
5
Q
What is a risk assessment?
A
A risk assessment needs to be carried out before any practical work in the lab.
6
Q
What do risk assessments involve?
A
Identifying hazards, risks and control measures to minimise the risk.
7
Q
Describe some control measures that can be put in place to reduce the risk.
A
Using appropriate handling techniques
Protective clothing and equipment (PPE)
Aseptic technique
8
Q
What is PPE?
A
Personal Protective Equipment
9
Q
Why is PPE worn?
A
PPE is worn to reduce the risk of harmful substances coming into contact with the body.
10
Q
Why are biological systems put into solution?
A
They are frequently put into solution for analysis.
11
Q
What do solution allow you to do?
A
Solutions allow you to transfer parts for sampling and can be diluted so you can better analyse.
12
Q
When working with liquids and solutions what should be considered when choosing apparatus?
A
Accuracy
Precision
The volumes used
13
Q
What would you use to make a really large dilution?
A
Log dilution series
14
Q
What would you use to make a small dilution?
A
Linear dilution series
15
Q
Describe a linear dilution series?
A
Dilutions in a linear dilution series differ by an equal interval, e.g. 0.1, 0.2, 0.3, and so on.
16
Q
How is a linear dilution series achieved?
A
This is usually achieved by using different volumes of the same stock solution and combining with different volumes of a suitable solvent, e.g. water.
17
Q
Describe the formula C1V1=C2V2
A
Use the formula C1V1 = C2V2
C1 = stock concentration
V1 = volume of stock required
C2 = concentration required for working solution
V2 = volume required of working solution
18
Q
Describe a Log dilution series.
A
Dilutions in a log dilution series differ by a constant proportion, e.g. 10-1, 10-2, 10-3, and so on.
19
Q
How is a Log dilution series is achieved?
A
This is usually achieved by using successive dilutions as the new stock solution.
20
Q
When are log dilution series useful?
A
Are particularly useful when diluting bacterial suspensions.
A large scale dilution is usually required to obtain countable numbers of colonies.
Often a range of dilutions are made and then plated out onto nutrient agar plates until a countable number of colonies is achieved.
21
Q
What is a standard curve used to determine?
A
The concentration of a solution.
22
Q
Describe a standard curve.
A
For a standard curve, a series of “standards” of known concentration are measured and graphed.
This graph can then be used to determine the concentration of an unknown solution.
23
Q
What is a titration used to determine?
A
Titrations can be used to determine the concentration of an unknown solution.
24
Q
Describe how to do a titration.
A
Titrations are carried out using a burette to deliver a solution of known volume and concentration to a solution of unknown concentration underneath.
The volume and concentration of the known solution taken to reach the endpoint of the reaction are used to calculate the concentration of the unknown solution.
25
Describe a Buffer.
Buffers are an important addition to an in vitro experiment, e.g. during an enzyme assay. It allows the pH of the reaction mixture to be kept constant (minimising pH as a confounding variable) because the addition of acid or alkali has very small effects on the pH of a buffer
26
What experiment is a Buffer useful in?
In Vitro
27
Describe how a colorimeter works.
It works by passing a light beam, at a specific wavelength, through a cuvette containing a sample.
Some of the light is absorbed by the sample, light transmitted is detected and displayed as an absorbance value.
28
What does a colorimeter measure?
A colorimeter measures the absorbance or transmission of light through a solution.
29
What can we determine by measuring the absorbance of light by a sample?
By measuring absorbance of light by a sample, we can determine the concentration of a coloured substance using suitable wavelength filters.
30
What is the relationship is there between absorbance and concentration?
A linear relationship between absorbance and concentration.
31
By measuring percentage transmission of light through a sample, what can we measure?
We can measure turbidity of a sample.
32
A colorimeter is used when calculating the cells in a suspension in microbiology work. What is the relationship between transmission and turbidity?
The lower the transmission, the higher the turbidity.
33
Tell me how to set up a colorimeter
To use a colorimeter the correct wavelength of light must be selected
An appropriate reference blank is required to calibrate the instrument and provide a baseline reading.
This is usually distilled water but depends on the reaction being investigated. The references should show you the absorbance in the absence of your coloured product.
The blank is placed in the colorimeter and the calibration button pressed (usually the R button). This will give a reading of 0.
Then sample cuvettes can be used by pressing the test button (T)
34
Biological systems often involve mixtures, name a few types of mixtures
Cells, DNA fragments, proteins and other compounds.
35
What can biological mixtures be separated based on?
These can be separated based on differences between the component parts. This can be differences in; solubility, size, shape or charge, or indeed any combination of these attributes.
36
What is a centrifuge?
A centrifuge is a piece of equipment that spins a sample at high speed.
37
What does centrifugation separate materials based on?
Density
38
Describe what happens in a centrifuge once it stops, where do the different densities of material go?
The largest and densest materials separate out first and form a pellet at the bottom of the tube. The less dense components remain in the supernatant.
39
What are paper and thin layer chromatography used for?
Are used for separating different substances such as amino acids and sugars. The speed that each solute travels along the chromatogram depends on its differing solubility in the solvent used.
40
What is paper chromatography?
Uses paper as the stationary phase and the solvent as mobile phase.
41
What is thin layer chromatography?
Uses cellulose or silica gel on a glass plate as stationary phase
42
Describe Affinity chromatography
Is used to separate proteins.
A solid matrix or gel column is created with specific molecules bound to the matrix or gel.
Soluble, target proteins in a mixture, with a high affinity for these molecules.
The protein mixture is passed through the column.
The target proteins become attached to the molecules.
Other non-target molecules with a weaker affinity are washed out.
43
What is Gel electrophoresis used to do?
Is used to separate proteins and nucleic acids.
44
What does Gel Electrophoresis separate molecules based on?
This process separates proteins based upon their charge and/or size/shape.
45
Describe Gel Electrophoresis
Protein electrophoresis uses current flowing through a buffer to separate proteins. The gel used in protein electrophoresis acts as a sieve, separating the proteins.
46
Describe SPD page electrophoresis
SDS-PAGE electrophoresis, this procedure denatures proteins and they are given a uniform negative charge.
This means that the proteins can be separated based on their size alone.
Small proteins travel further through the gel than large proteins.
47
Describe Native Gel electrophoresis.
Native gel electrophoresis is when the protein is not denatured before the gel electrophoresis. This allows the scientist to analyse the proteins in their folded state. In this case, separation is by shape, size and charge.
48
What is the Isoelectric point?
The IEP is the pH at which a soluble protein has no net charge and will precipitate out of solution.
49
Describe the use of the Isoelectric point in separating proteins?
If the solution is buffered to a specific pH, only the protein(s) that have an IEP of that pH will precipitate.
Proteins can also be separated using their IEPs in electrophoresis.
Soluble proteins can be separated using an electric field and a pH gradient.
A protein stops migrating through the gel at its IEP in the pH gradient because it has no net charge.
50
What are Immunoassay techniques used to detect and identify?
Immunoassay techniques are used to detect and identify specific proteins.
51
Immunoassay techniques use to stocks antibodies with what to recognizes one antigen?
These techniques use stocks of antibodies with the same specificity, recognises one antigen.
52
hese techniques use stocks of antibodies with the same specificity, recognises one antigen.
What are these antibodies known as?
Monoclonal antibodies.
53
Describe Immunoassay techniques.
Immunoassay techniques are used to detect and identify specific proteins.
These techniques use stocks of antibodies with the same specificity, recognises one antigen.
These antibodies are known as monoclonal antibodies. An antibody specific to the protein antigen is linked to a chemical ‘label’ to allow scientists to detect when binding has occurred.
The ‘label’ is often a reporter enzyme producing a colour change, but chemiluminescence, fluorescence and other reporters can be used. In some cases the assay uses a specific antigen to detect the presence of the antibodies.
54
Bright-field microscopy is commonly used for what?
Bright-field microscopy is commonly used to observe whole organisms, parts of organisms, thin sections of dissected tissue or individual cells.
55
What does Fluorescence microscopy use?
Fluorescence microscopy uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues.
56
What does aseptic techique do?
Aseptic technique eliminates unwanted microbial contaminants when culturing micro-organisms or cells.
57
What does aseptic technique involve?
This involves the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contaminants.
Sterilising benches with virkon
When opening bottles – the neck must be immediately passed through a hot flame
Complete work close to a Bunsen burner flame where air currents are drawn upwards.
When using petri dishes, limit exposure of the sterile inner surface to contamination from air.
58
A microbial culture can be started using what?
A microbial culture can be started using an inoculum (a small volume) of microbial cells on an agar medium, or in a broth with suitable nutrients.
59
What exists to promote the growth of specific types of cells and microbes.
Culture media
60
What are animal cells grown in?
Medium containing growth factors from serum.
61
What are the growth factors?
Growth factors are proteins that promote cell growth and proliferation. Growth factors are essential for the culture of most animal cells.
62
What's the difference between primary cell lines and tumour cell lines?
In culture, primary cell lines can divide a limited number of times, whereas tumour cells lines can perform unlimited divisions.
63
Where do primary cell lines come from?
Primary cell lines – are isolated from human or animal tissue
64
Plating out of a liquid microbial culture on solid media allows what to be estimated?
Allows the number of colony-forming units to be counted and the density of cells in the culture estimated.
65
What is often needed to achieve a suitable colony count?
Serial dilution
66
what does a haemocytometer allow an estimation of?
A haemocytometer allows an estimation of the number of cells in a liquid culture.
67
what does a haemocytometer resemble?
It resembles a microscope slide, but has a grid made up of perpendicular lines (similar to graph paper) etched into the glass.
68
A haemocytometer can then be used to estimate both the total number of cells and the number of what?
A haemocytometer can then be used to estimate both the total number of cells in culture and the number of viable cells.
69
In a haemocytometer estimates of viable and total cell counts can be made using what? Which is absorbed by what?
trypan blue dye, which is absorbed by dead cells
70
Describe Vital Staining.
Vital staining is required to identify and count viable cells.
It involves adding a stain to a cell culture and then observing which cells have taken up the stain.
71
What is the proteome?
The proteome is the entire set of proteins expressed by a genome.
72
The proteome is larger than the number of genes (particularly in eukaryotes)due to what?
Alternative RNA splicing (more than one protein can be produced from a single gene.
73
Describe RNA Splicing.
Introns are the non-coding sequence of the mRNA and will not be expressed in the protein molecule. They are spliced out (removed) from the mRNA. The mRNA can then leave the nucleus via a nuclear pore and enter the cytoplasm.
Exons are the coding sequence and will be expressed in the protein molecule.
74
Not all genes are expressed as proteins in a particular cell type.
Genes that do not code for proteins are called what?
Non-coding RNA genes
75
Name some roles of Non-coding RNA genes.
transcribed to produce tRNA, rRNA and RNA molecules that control the expression of other genes.
76
The set of proteins expressed by a given cell type can vary over time and under different conditions.
Name these conditions which cause variation.
Metabolic activity of the cell
Cellular stress
Response to signalling molecules
Diseased versus healthy cells.
77
Describe the Proteome.
The proteome is larger than the genome due to RNA splicing and post-translational modification. As a result of gene expression not all genes are expressed as proteins in a particular cell.
78
Because of their size, eukaryotes have a relatively small what to what ratio
surface area to volume ratio.
79
The plasma membrane of eukaryotic cells is too small an area to carry out what?
all the vital functions carried out by membranes.
80
Eukaryotic cells have a system of internal membranes, what does this increase?
The total area of membrane available for cellular functions.
81
Describe the endoplasmic reticulum
The endoplasmic reticulum (ER) forms a network of membrane tubules continuous with the nuclear .membrane.
82
What does the endoplasmic reticulum do?
It is involved in the synthesis of proteins and lipids.
83
Describe the 2 types of Endoplasmic reticulum.
The ER is called the rough ER (RER) when ribosomes are on its cytosolic face.
If the ER lacks ribosomes then it is called the smooth ER (SER).
84
Describe the Golgi apparatus.
The Golgi apparatus is a series of flattened membrane discs.
85
What does the Golgi apparatus do?
It is involved in the transport and modification of proteins.
86
What are Lysosomes?
Lysosomes are membrane-bound organelles containing a variety of hydrolases that digest proteins, lipids, nucleic acids and carbohydrates.
87
What do vesicles do?
Vesicles transport materials between membrane compartments.
88
Where are lipids synthesised?
Lipids are synthesised in the smooth endoplasmic reticulum (SER) and inserted into its membrane.
89
The synthesis of all proteins begins where?
Cytosolic ribosomes
90
The synthesis of cytosolic proteins is completed in the cytosolic ribosomes, and where do these proteins remain?
The synthesis of cytosolic proteins is completed there (cytosolic ribosomes), and these proteins remain in the cytosol.
91
Transmembrane proteins carry a signal sequence, which does what?
Halts translation and directs the ribosome synthesising the protein to dock with the ER, forming RER (rough endoplasmic reticulum)
92
What is a signal sequence?
A signal sequence is a short stretch of amino acids at one end of the polypeptide that determines the eventual location of a protein in a cell.
93
Translation continues after docking, where is the protein inserted into?
the membrane of the ER.
94
Once the proteins are in the ER, they are transported by what that bud off from the ER and fuse with the Golgi apparatus?
Vesicles
95
As proteins move through the Golgi apparatus they undergo what?
Post-translational modification.
96
Name an example of post-translational modification that happens in the Golgi apparatus.
The addition of carbohydrate groups to proteins is the major modification taking place in the Golgi. Enzymes catalyse the addition of various sugars in multiple steps to form the carbohydrates.
97
Describe the movement of molecules through the Golgi apparatus.
Molecules move through the Golgi discs in vesicles that bud off from one disc and fuse to the next one in the stack.
Vesicles that leave the Golgi apparatus take proteins to
the plasma membrane and lysosomes.
Vesicles move along microtubules to other membranes and fuse with them within the cell.
98
Secreted proteins (such as peptide hormones and digestive enzymes) are translated in where and enter what on it?
Ribosomes on the RER and enter its lumen.
99
How do proteins get out of the cell?
The proteins move through the Golgi apparatus and are then packaged into secretory vesicles. These vesicles move to and fuse with the plasma membrane, releasing the proteins out of the cell.
100
What is proteolytic cleavage
Proteolytic cleavage is the process of breaking the peptide bonds between amino acids in proteins. This is another type of post-translation modification.
101
Many secreted proteins are synthesised as inactive precursors and require proteolytic cleavage to produce active proteins. Name an example.
For example, this prevents digestive enzymes becoming active in an inappropriate location and causing damage to the cell.
102
Proteins are polymers of what?
Amino acid monomers.
103
Amino acids are linked by what to form polypeptides
Peptide bonds
104
What is a peptide bond made up of
The –OH from one amino acid bonds to the -H from the -NH2 group on another amino acid.
105
Amino acids have the same basic structure, differing only in what?
The R group present.
106
What can amino acids vary in?
These can vary in size, shape, charge, hydrogen bonding capacity and chemical reactivity.
107
What are the 4 different type of R group
Acidic (negatively charged), Basic (positively charged), polar and hydrophobic (nonpolar)
108
Name a basic amino acids name?
Lysine (CH2)4NH2
109
Name an acidic amino acid name?
Aspartic acid
(asp)-
Carboxyl group
(CH2)COOH
```
Glutamic acid (glu)
Carboxyl group (CH2)2COOH
```
110
Name a polar amino acid.
Serine(ser)
CH2OH
Asparagine
(asn)
CH2CONH2
111
Name a hydrophobic amino acid.
```
Glycine
Gly
Alanine
Ala
Cysteine
Cys
```
112
Name the 4 levels of protein structure
Primary -Amino acid sequence
Secondary - Regular sub structures (alpha helix or beta sheets)
Tertiary - 3-dimensional structure
Quarternary - Complex of protein molecules
113
Describe the primary structure of an amino acid.
The primary structure is simply the sequence in which the amino acids are synthesised into a polypeptide
114
Describe the secondary structure of an amino acid.
Hydrogen bonding along the backbone of the protein strand results in regions of secondary structure.
There are 3 types of secondary structure α-helix, parallel or anti-parallel β–sheet and turns.
115
Describe the tertiary structure of an amino acid
Tertiary structure involves the folding of the polypeptide chains to give a more complex 3D structure.
This conformation is stabilised by interactions between the R groups
116
In tertiary structure what types of interactions take place?
```
Interactions between R groups can be:
Hydrophobic interactions
Ionic bonds
London dispersion forces
Hydrogen bonds
Disulfide bridges
```
117
What are disulfide bridges?
Disulfide bridges are covalent bonds formed between R groups containing sulphur.
118
Describe the quaternary structure of amino acid.
Quaternary structure exists in proteins with two or more connected polypeptide subunits.
This describes the spatial arrangements of the subunits.
119
What are prosthetic groups?
Prosthetic groups are non-protein unit tightly bound to a protein and necessary for its function
120
Hemoglobin is an example of the quaternary structure of amino acid. The ability of hemoglobin to bind oxygen is dependent upon what?
The non-protein haem group.
121
Increasing temperature does what to influence the interactions of the R groups?
Increasing temperature disrupts the interactions that hold the protein in shape. The protein begins to unfold, eventually becoming denatured.
122
The charges on acidic and basic R groups are affected by pH, describe what happens.
As pH increases or decreases from the optimum, the normal ionic interactions between charged groups are lost. This gradually changes the conformation of the protein until it becomes denatured.
123
What is a ligand?
A ligand is a substance that can bind to a protein.
124
Describe the binding of a ligand in as much detail as possible
R groups not involved in protein folding can allow binding to ligands.
Binding sites will have complementary shape and chemistry to the ligand.
The ligand can either be a substrate or a molecule that affects the activity of the protein.
125
Describe the conformation changes that happen when a ligand binds
As a ligand binds to a protein-binding site, or a substrate binds to an enzyme’s active site, the conformation of the protein changes.
This conformational change causes a functional change in the protein (may activate or deactivate the protein).
126
Allosteric interactions occur between what?
Spatially distant sites. Allo’ means ‘other’ as in ‘other than the active site’.
127
Many allosteric proteins consist of multiple subunits.
What type of protein structure is this?
Quaternary
128
Describe cooperativity
Allosteric proteins with multiple subunits show co-operativity in binding.
The binding of a substance molecule to one active site of an allosteric enzyme increases the affinity of the other active sites for binding of subsequent substrate molecules.
129
WHY IS COOPERATIVITY OF BIOLOGICAL IMPORTANCE
This is of biological importance because the activity of allosteric enzymes can vary greatly with small changes in substrate concentration.
130
Define affinity
The attractive force binding atoms in molecules; the tendency to combine and form bonds in a chemical reaction
131
Allosteric enzymes contain a second type of site, called what?
An allosteric site.
132
What do modulators do?
Modulators regulate the activity of the enzyme when they bind to the allosteric site.
133
Following the binding of a modulator what happens
Following binding of the modulator, the conformation of the enzyme changes and this alters the affinity of the active site for the substrate.
134
What do positive modulators do?
Positive modulators increase the enzyme’s affinity for the substrate.
135
What do negative modulators do?
Negative modulators decrease the enzyme’s affinity for the substrate.
136
The binding and release of what in haemoglobin shows co-operativity.
oxygen
137
Describe cooperativity in haemoglobin
Haemoglobin has a relatively low affinity for oxygen.
As one molecule of oxygen binds to one of the four haem groups in a haemoglobin molecule it increases the affinity of the remaining three haem groups to bind oxygen (makes the binding of other oxygen molecules more likely).
138
Describe the factors that affect the binding of oxygen to hemoglobin
The main factors which affect haemoglobin's ability to bind oxygen are:
temperature - as temperature increases, affinity of haemoglobin for oxygen decreases;
pH - as pH decreases, affinity of haemoglobin for oxygen decreases.
139
What is the Phosphorylation of proteins a form of?
Phosphorylation of proteins is a form of post-translational modification.
140
The addition or removal of phosphate from particular R groups can be used to cause what?
Reversible conformational changes in proteins.
141
What is Phosphorylation?
adding a phosphate
142
What is dephosphorylation?
removing a phosphate
143
What does the protein kinase do?
Protein kinases catalyse the transfer of a phosphate group to other proteins.
The terminal phosphate of ATP is transferred to specific R groups.
144
What is phosphorylation ?
Adding a phosphate adds a negative charges to the protein.
| This can disrupt ionic interactions in the unphosphorylated protein and create new ones.
145
What is phosphatase?
Protein phosphatases catalyse dephosphorylation of other proteins by the removal of phosphate from the protein molecule.
This again changes the conformation of the protein as a result of charge interactions of the R groups in the protein.
146
Where does the phosphate come from.
ATP
147
What does dephosphorylation/ phosphorylation do?
Phosphorylation/dephosphorylation allows the activity of many cellular proteins, such as enzymes and receptors, to be regulated.
148
Some proteins are activated by what while others are inhibited.
Phosphorylation
