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Introduction to Laboratory Science > Blood and Saliva > Flashcards

Blood and Saliva Flashcards

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1
Q

What risk procedures should you take with this practical?

A

Wear goggles and gloves to protect skin and eyes

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2
Q

What is each pipette named by?

A

It’s maximum volume it can pipette

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3
Q

How do you set the volume on a pipette?

A
  • Use the thumb wheel to set the volume on the dial
  • The red number shows decimals
  • For the P1000 100 indicates 1000 ul and 052 would indicate 520 ul
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4
Q

What volume should you never pipette with a certain pipette?

A

A volume outside it’s range

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5
Q

What should you know do with pipette tips?

A
  • Once you have set the volume attach the appropriate pipette tip
  • P20, P50 and P200 pipettes use the same yellow tip
  • P1000 pipettes use a larger blue tip
  • P10 uses a white tip
  • A filter tip is often used as well.
  • discard of tip once used
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6
Q

What is the pipetting procedure?

A
  • Liquid drawn in and propelled using pipette’s push button that is
  • Gently push until you get to the first stop
  • Then place the pipette tip about 2mm into the liquid
  • Slowly release the push button and pause for a second
  • Place the pipette into the recipient container and push the push button to the second stop
  • Remove the pipette from the liquid without releasing the push button
  • For very small volumes press the pipette tip to the side of the wall to release it
  • After finished with the tip release the pipette tip into the waste container by pressing on the tip ejector (small white button that is not the push button)
  • A new tip should be used for each new liquid
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7
Q

What are common things to avoid when pipetting?

A
  • Don’t use a pipette without a tip attached
  • Don’t use a pipette past it’s volume limits
  • When drawing up the liquid don’t press past the first stop
  • Make sure to release the push button in a controlled manner
  • Make sure the tip stays below the surface of liquid
  • Don’t lay the pipette down when you have liquid in it
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8
Q

What is spectrophotometry used to do?

A

identify compounds (qualitative) and their concentration (quantitative) based on the specific wavelengths of light they absorb

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9
Q

What is the principle of spectrophotometry?

A
  • When white light strikes an object, different wavelengths (colours) may be reflected, transmitted or absorbed depending on the structure of the molecules within the object
  • When molecules are in a solution, we can detect the wavelengths transmitted and the wavelengths absorbed
  • Spectrophotometry is a technique used to identify a molecule by the wavelengths (UV or colour in visible light) it transmits and absorbs
  • The spectrophotometer can detect the wavelengths transmitted, deduce those absorbed and return an absorption spectrum
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10
Q

Why will only certain wavelengths of light be absorbed by a molecule depending on it’s structure?

A
  • When UV or visible light is absorbed by a compound the outer (valence) electrons are excited from the lowest energy state, the ground state to a higher energy level, the excited state
  • There is a fixed value for the energy required for a particular electronic transition. Therefore only certain wavelengths of light will be absorbed by a molecule, depending on it’s structure.
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11
Q

What are conjugated bonds and what does this mean for molecules with conjugated bonds?

A
  • conjugated bonds are when single and double bonds alternate and the bonds combine and the electrons are delocalised over all the atoms
  • the presence of conjugated bonds lowers the energy required for an electronic transition
  • therefore molecules with conjugated bonds may be able to absorb light in the UV and/or visible spectrum
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12
Q

What is the basic set up of a spectrophotometer?

A
  • Light source
  • Goes through collimator
  • Goes through monochromator
  • Goes through slit
  • Goes through sample
  • Is detected
  • Readout
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13
Q

What’s the light source and which light sources are used when?

A
  • Can be a tungsten lamp which emits light in the visible spectrum (340-800nm) so can be used to explore coloured molecules
  • A deuterium lamp can also be used which emits light in the UV region (200-340nm) as some colourless molecules such as DNA absorbs light in the UV range
  • Both lamps are used when a molecule absorbs light in both ranges (e.g. haemoglobin)
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14
Q

What is the collimator?

A

a lens that directs the light from the light source into a beam. It then focuses this beam onto a monochromator

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15
Q

What does light passing through the monochromator do?

A

form a spectrum and light of a particular wavelength is produced

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16
Q

What is a slit used to do?

A

to ensure only the desired wavelengths of light are transmitted through the sample

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17
Q

What does a detector do?

A

measures the intensity of light transmitted through the sample and from this computes the light absorbed by the sample

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18
Q

Where is the readout presented?

A

on a digital display

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19
Q

What does a spectrophotometer measure to measure the amount of light absorbed by a sample at a particular wavelength?

A
  • The intensity of light entering the solution

- The intensity of light leaving the solution

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20
Q

What is the equation to calculate absorbance?

A

log I0/It

  • I0 is the initial intensity
  • It is the intensity of the light leaving the solution
  • absorbance is the ration between Io and It so there are no units
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21
Q

How is I0 measured practically?

A

Using a blank cuvette/ reference

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22
Q

What is the blank solution and why is it required?

A
  • The blank solution is identical to the sample solution but does not contain the solute that absorbs the light. Usually water or a buffer is used. This reference is required because the cuvette itself and the solvent may absorb and/or scatter some of the light
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23
Q

what does the absorbance at different wavelengths of light depend on?

A

the molecular structure of the solute

24
Q

What is an absorbance spectrum and what can it be used to do?

A
  • graph of absorbance vs wavelength

- can be used to identify the molecule within a sample because it will have a specific absorbance spectrum

25
Q

What is the wavelength at which the absorbance is the greatest known as?

A

the lambda max

26
Q

What is the absorbance of the sample proportional to?

A

The concentration of the sample

27
Q

What does the Beer-Lambert law describe?

A

the linear relationship between absorbance (A) and the molar concentration (C) of an absorbing species at a particular wavelength

28
Q

What is the beer-lambert equation?

A

A = εlc (with the lambda sign before them)

  • A is the absorbance
  • ε is the molar extinction coefficient (This is constant for a specific compound at a particular wavelength) also known as epsilon.
  • l is the length of the path the light must travel in the solution (the length of the cuvette, usually 1cm)
  • c is the molar concentration of a given solution in M or mol l^-1 (mol per litre)
29
Q

What does the beer lambert law relate to and so what would you expect with a graph of concentration against absorbance?

A

the equation of a straight line: y = ax + b

  • Therefore if a graph of concentration against absorbance was plotted, you would expect a straight line which passes through the origin
  • ε is equivalent to the gradient of the line
30
Q

How can concentration be worked out if ε is known?

A

by measuring absorbance and rearranging the Beer Lambert equation

31
Q

What is a cuvette?

A
  • a small (-3mL) plastic tube

- made of plastic quartz or glass and is usually 1 cm wide

32
Q

What are key points to remember about cuvette handling?

A
  • A cuvette is easily knocked over so keep it in the rack of the spectrophotometer
  • When you handle the cuvette, avoid touching the light path as you could add grease or fingerprints which will obstruct the reading
  • Typically handle the cuvette via its top edges but you can also touch the bottom part
  • Reuse the cuvette by rinsing it between uses with distilled water and tapping it upside down on blue roll to clear excess water
  • If there are any air bubbles it could obstruct the reading (scatter the light) so gently tap the cuvette to remove any air bubbles
  • If the side of the cuvette is wet, wipe it with a tissue
  • Do not overfill the cuvette or place it on top of the machines as the solution may spill.
33
Q

How do you generate an absorbance spectrum?

A
  1. Open the lid of the spectrophotometer and place the blank cuvette in the cell holder. Make sure the cuvette is in front of the lamp window and then close the lid
  2. touch spectrum on the home screen
    - use the touch screen to adjust the parameters
    - you will need to measure the absorbance between a set range
  3. Then touch the forward arrow and a new screen will appear. First the machine must establish the baseline for the reference (blank) sample which it will automatically subtract from the absorption spectrum later
  4. press blank
  5. empty the blank from the cuvette, rinse and tap and transfer the light absorbing solution into the cuvette and place the cuvette back in the machine
  6. touch ‘read’ and a graph will appear below. Note the corresponding wavelength for the maximum absorbance
  7. take a picture and sketch the spectrum taking note of the wavelengths on top of each peak as these peak wavelengths are the signature of your molecules
34
Q

What is the absorbance spectrum for Hb?

A
  • The peak at 273 nm is broad and correspond to different amino acids absorbing UV light
  • The sharp peak at 406nm comes from the haem group which gives the brown colour to our solution
35
Q

What is the absorbance spectrum of theatrical blood?

A

Made up of sugar (260nm: colourless) and three colorants: yellow (327nm), orange (around 450nm) and red (514 nm)

36
Q

How do you work out the dilution factor?

A

total volume/initial volume of (stock)

37
Q

What are the properties of native proteins?

A

• Proteins in all species are constructed from the same set of twenty amino acids
• Each protein has a unique amino acid sequence determined by the order of nucleotide bases in its encoding gene
• Differences in their chemical properties such as charge, shape, size and solubility enable them to perform many biological functions, including enzyme catalysis, metabolic regulation, binding and transport of small molecules, gene regulation, immunological defence and cell structure.
• Amino acids are made of an amino group, a carboxyl group and an R group
• Amino acids are linked together to form a polypeptide chain
• The unique chemical composition of each R group is what gives each amino acid its important characteristics such as chemical reactivity, ionic charge and relative hydrophobicity
- Each amino acid has a positive, negative or neutral charge so the net charge of each protein is naturally different
- Each amino acid provides different instructions for the tertiary and quaternary structures of the protein, so the 3D shape of each protein is naturally different

38
Q

What are the different structures of proteins?

A
  • Primary structure: linear arrangement of amino acids linked by peptide bonds
  • Secondary structure: folding or coiling through alpha helices and pleated sheets, which are stabilized by hydrogen bonds
  • Tertiary structure: the final 3D structure resulting from interactions between atoms of the proteins and the environment. Covalent disulphide bridges hold the folded polypeptides, hydrophobic residues in amino acids gather away from water to create folds stabilised by Van de Waals forces and ionic bonds form between positive and negative charges
  • Quaternary structure: Stems from non-covalent interactions that bind multiple polypeptides into a single, larger protein. Haemoglobin has a quaternary structure due to the association of two alpha globin and two beta globin polyproteins
39
Q

What is gel electrophoresis?

A

The utilisation of a gel matrix and an applied electric field to separate molecules (DNA,RNA or proteins) by their physical or chemical properties (size, shape and electric charge)

40
Q

What is the basic principle of gel electrophoresis?

A
  • The gel matrix acts as a molecular sieve, such that smaller molecules move through it more quickly than larger molecules
  • The degree of sieving can be controlled by selecting the appropriate gel substance – agarose for large molecules (DNA); polyacrylamide for smaller molecules (proteins or a small nucleic acid)
  • By changing the concentration of the gel matrix: the higher the percentage of agarose or polyacrylamide, the denser the matrix
  • The electrodes at the top and bottom of the gel influence the migration of the molecules through the matrix according to their charge
41
Q

What are the two types of PAGE electrophoresis for proteins?

A
  • Native PAGE where the proteins are intact and separated on the basis of their size, shape and charge
  • SDS-PAGE where the proteins are denatured and negatively charged and separated on the basis of size only
42
Q

How do proteins of the same size migrate in native PAGE?

A

migrate differently, even if only different by one amino acid, because the change will affect their shape and/or charge

43
Q

How do proteins of the same size migrate in SDS-PAGE?

A

proteins of the same size migrate to the same position in the gel even if made of different amino acids

44
Q

How does SDS-PAGE electrophoresis work?

A
  • It has a polyacrylamide gel matrix. Acrylamide forms linear polymers and bisacrylamide introduces crosslinks between polyacrylamide chains, creating a network of tunnels. Their diameter (sieving power of the matrix) is determined by the ratio of acrylamide to bisacrylamide and by the concentration of acrylamide
  • Samples containing proteins are loaded in wells and when an electric field is applied, protein molecules migrate in a direction and at a speed that reflects their net size and charge
45
Q

What is SDS used to do?

A

solubilise proteins and coat them with negative charges so that they migrate towards the anode through the polyacrylamide gel matrix. Denaturation of proteins is made complete by using heat and a reducing agent that breaks the disulphide bonds in or between proteins. Under these conditions the proteins migrate at a rate that reflects their molecular weight only

46
Q

What is the principle of SDS?

A
  • To determine a protein’s molecular weight or separate proteins according to size only mass must be the only factor determining the migration rate
  • This means that the m/z ratios (mass to charge) of all protein molecules must be equalised and the proteins must be denatured back to their primary structure
47
Q

How are proteins denatured to their primary structure?

A

heating the proteins with chemical reagents present in both the sample loading buffer (called the laemmli buffer) and the gel running buffer

  1. Heat disrupts hydrophobic interactions and dipole-dipole interactions
  2. A thiol reducing agent disrupts any disulphide bonds
  3. The anionic detergent sodium dodecyl sulphate (SDS) solubilises, denatures and coats the proteins with negative charges
48
Q

What are the properties of SDS that ensures that the proteins will be solubilised and coated with negative charges?

A
  • SDS is a detergent that has a lipophilic tail, and so can dissolve hydrophobic molecules. This property ensures that when a cell in incubated with SDS, the membranes are dissolved and the proteins are solubilised by the detergent
  • SDS also has an anionic head group (sulphate). By binding non-covalently to proteins with a stoichiometry of around 1 SDS molecule per 2 amino acids, this property ensures that all proteins, regardless of their intrinsic net charge will be similarly covered with negative charges
49
Q

How is the migration rate monitored while the gel is running considering that proteins in the sample are not visible when the gel is running?

A

a Bromophenol Blue dye is mixed with the protein samples. The dye molecules are negatively charged and are smaller than the proteins expected in most dye samples, so they constitute the leading front. When the dye approaches the bottom of the gel it is time to turn off the power.

50
Q

How are proteins made visible after the gel is run?

A
  • the gel is placed into the Coomassie blue dye.
  • This dye binds specifically to proteins and not other macromolecules
  • after destaining and getting rid of the unwanted blue dye, distinct blue bands will appear on the gel revealing where the proteins are.
  • The amount of dye binding to the proteins is directly proportional to the amount of proteins present
51
Q

What are molecular weight markers and what are they used for?

A
  • used in order to determine the size of unknown proteins
  • they are proteins of known size which run along-side our sample on the SDS page
  • consist of a mixture of proteins of known molecular weight
  • either unstained or prestained with covalently attached dyes
52
Q

What is the relationship between the migration rate of a protein coated with SDS and the logarithm of it’s molecular weight?

A

Inversely proportional

y = a + bx
y - log molecular weight (kDA)
x - distance migrated in mm

53
Q

How do you determine the molecular weight of the unknown bands on the gel?

A

plot Y = log of MW (in kDA) of your molecular weight markers versus X = distance (in mm) the molecular weight markers have migrated from the bottom of the well into the SDS-PAGE gel

54
Q

What is 1% (w/v)?

A

1 g/100 ml

55
Q

What does the intensity of a band reflect?

A
  • The quantity of proteins present in that band

- the darker the band the more proteins it contains and it often gets a bit thicker too

Introduction to Laboratory Science (5 decks)

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