MiMS practical notes

Serial dilution

When making a serial dilution for a factor of 10, the parts have to add up to 10 within the total volume required, e.g. if you want 1ml at a dilution of 10, then would be 100ul from the stock solution and 900ul water. If the stock solution was 300M then the dilution would be 30M. Repeat for subsequent dilutions.


Bradford assay

Used to estimate the concentration of protein in the sample by using the dye Coomassie Blue. When Coomassie blue binds to protein it is converted from an unstable, positively-charged form to a stable, negatively-charged form.

Components of Bradford reagent are coomassie blue dye and phosphoric acid.

Reference protein is BSA (Bovine Serum Albumin).

Colourimeter must first be calibrated with a blank, then standards (BSA of known concentrations) to generate a calibration curve. Then the mystery protein concentration sample can be added to a cuvette.

Before binding to the protein:

Colour = red/brown & max absorbance = 465 nm

After binding to the protein:

Colour = blue & max absorbance = 595 nm

Therefore colour change is measurable with a colourimeter - more protein bound to the dye = higher the absorbance.

Steps to generate the calibration curve:

  1. Add the Bradford reagent to the BSA standards.

  2. Mix to ensure the reaction initiates evenly throughout the sample.

  3. Leave for 5 mins to ensure that the reaction goes to completion and the colour is fixed.

  4. Blank the spectrophotometer so that there are no background readings.

  5. Measure the absorbance.

  6. Plot a graph of absorbance (y-axis) against protein concentration (x-axis).

NOTE - When plotting the curve and choosing the standards, ensure that the sample concentrations (even though these are a mystery so I do not know how one would do this) fall within the lower-middle regions of the absorbance curve, as it starts to plateau at high concentrations and small errors have a large effect at low concentrations.

For linear calibration curves - either the curve or the Beer-Lambert law can be used.

For variable calibration curves - the curve should be used.


SDS-PAGE

A lab technique that separates proteins by size. Used to assess the purity of a sample or identify proteins within a sample.

SDS: Sodium dodecyl sulfate. Coats the proteins in a uniform, even negative charge.

PAG: Polyacrilimide gel. A polymer mesh that the proteins have to physically move through.

E: Electrophoresis. The separation of charged particles through application of an electrical voltage.

Protein is also heat-treated after the addition of SDS to prevent it refolding.

Smallest proteins are the least restricted by the gel mesh so they travel further once the voltage is applied than the larger proteins.

Ladder proteins (proteins of known sizes) are added to the gel alongside the samples so that the sizes of the sample proteins can be known.

Voltage between 40-160V is used. Too low = fuzzy, diffuse bands, too high = melted gel.

After the voltage has run, the proteins need to be stained to be visible, as only the dye front can be seen. After the gel has been stained, it must be destained to remove the excess dye from the gel. Often multiple rounds of destaining are needed to ensure that the protein bands can be seen against the gel background.


Western blot

Used to detect a specific protein in a mix of other proteins. Includes an SDS page but then protein bands undergo immunoblotting with antibodies.

  1. Carry out an SDS page to separate the proteins by their molecular weights.

  2. Transfer the protein bands (elution) onto a porous membrane.

  3. Primary antibodies are added. These stick to the protein of interest. Wash.

  4. Secondary antibodies attached to an enzyme are added. These stick to the primary antibodies. Wash.

  5. Enzyme substrate is added. If the enzyme breaks down the substrate it will produce light, which is then detected so the location of where the protein is bound can be found.


Before adding the antibodies it is important to add a blocking solution beforehand (proteins that bind to the areas of the blot not occupied by the sample) to prevent antibodies binding to places other than the membrane.

Secondary antibody must be specific to the host species in which the primary antibody was generated, but must not be raised in that species itself, i.e. if primary antibody is rabbit, then secondary antibody cannot be rabbit anti-rabbit, it could be e.g. mouse anti-rabbit or goat anti-rabbit.

Loading control is a sample where the protein concentration is known. Included when loading a Western Blot because it tells us that the same amount of sample was loaded into each well.

The presence of a band tells us the protein is present, and the size of the band tells us how much protein is present.


Sandwich ELISA

Used to detect and quantify the presence & concentration of a substrate using antibody/antigen interactions.

  1. Antibodies complementary to an antigen are immobilised via their constant region onto a plate. Wash.

  2. Blocking buffer is added to prevent non-specific binding of antigens/antibodies to other areas of the plate. Wash.

  3. Mixed protein sample is added. The antigen of interest becomes bound to the immobilised antibodies.

  4. Primary antibodies are added that detect and bind to the antigen. Wash.

  5. Secondary antibodies are added but these ones are attached to enzymes and are complementary to the constant region of the primary antibody. Note that multiple secondary antibodies can bind to one primary antibody - this amplifies the end signal. Wash.

  6. Enzyme substrate is added and a reaction occurs. Intensity of the colour change proportional to concentration. Detected using a spectrophotometer.

  7. Intensity of the signal is used to calculate the concentration of the antigen by comparing with a standard curve of (graph of absorbance vs concentration for a set of standards).

Standards, positive control & negative control should also be run.


Ion exchange chromatography

General rule is that increasing the pH (making solution more alkali) causes the charge on a protein to become more negative. Isoelectric point is when the overall charge of the protein is zero. pH less than isoelectric point protein = positive; greater than isoelectric point protein = negative.

Column with charged beads is set up and the pH selected by using a buffer of a certain pH. This means that proteins will either be eluted or bound to the beads depending on the relation of their isoelectric point & the buffer and the charge of the beads.

Cation exchange column = negatively charged beads

Anion exchange column = positively charged beads.


Gel filtration chromatography

Uses beads with pores of various sizes to separate the proteins by size. Large proteins cannot enter the gel pores so they travel rapidly and elute first. Small proteins diffuse through most of the pores, slowing them down so they elute last.


Michaelis-Menten plot

Describe the relationship between rate of an enzyme controlled reaction and the substrate concentration.

Estimate for vmax is the rate at which the graph will theoretically plateau. Km is the substrate concentration at 50% of the vmax.


Lineweaver-Burke plot

Used to find the Km and the vmax more accurately. x-intercept = -1/km; y-intercept = 1/vmax. Gradient = km/vmax. x axis = 1/[S]. y axis = 1/v


PCR

Used to amplify a region of DNA (amplicon).

  1. Heat the dsDNA to 90 to denature the DNA (break the hydrogen bonds between the bases).

  2. Temperature reduced to 50-60 to allow the primers to anneal. Primers are usually 20bp long. Made from dATP, dTTP, dCTP & dGTP.

  3. Taq polymerase binds to the primer. Temperature raised to 70. Extends the primers and synthesises the new strand.

  4. After 3 rounds of PCR there are double stranded versions of the amplicon without ssDNA overhangs.

  5. Repeat cycle 30-40 times.

  6. Products are analysed with gel electrophoresis.

Note that DNA copy number may not be equal to 2^number of cycles in practice because…

  • Taq polymerase may not completely synthesise each strand.

  • Molecules may become depleted as the cycles progress.

  • Not every strand will denature.

  • Taq polymerase & primers will not bind to every strand.

Also primers should not be complementary to each other, only to the DNA strand, otherwise they will bind together.

Positive (definitely contains the amplicon) and negative controls (definitely does not contain the amplicon) are also run alongside the samples.


Agarose gel electrophoresis

Used to separate strands of DNA according to their length. Longer DNA strands will travel a shorter distance than shorter DNA strands as they will be more impeded by the agarose gel matrix.

Buffer solution used when the electrophoresis is being run to keep the pH constant, the gel moist and to provide enough ions to carry an electrical current.

Loading buffer is added to the samples before loading.

Loading buffer = dense chemical (keeps sample in the wells), fluorescent dye (allows results to be visualised) & coloured dye (so that progress can be tracked as the gel is running).

DNA ladder is also added to a separate well so that the DNA fragment sizes can be found.

After gel electrophoresis has run, a transilluminator (uses UV light) is used to visualise the fluorescent dye in the loading buffer to reveal the DNA location in the gel.

Number of bands = number of different sized DNA fragments present.

Non-linear (logarithmic) difference between DNA length and distance.

DNA ladder can also be used to plot a standard curve between migration distance and log(DNA length).