Dit zijn de belangrijke dingen die in het pre-lab assignment zijn behandeld. Het gaat vooral over de verschillende methodes (SDS-page, western blot etc) die zijn gebruikt.
IgE must bind to two epitopes before an allergic reaction results. Besides, a conformational epitope
will be lost during heating step in the oven. This happens because the protein unfolds upon heating.
The tertiary structure consists of the three-dimensional structure of a protein, which can be
stabilized by S-S bridges. This is present within the molecule. The quaternary structure can also use
S-S bridges to form the spatial arrangements of multiple polypeptide chains. These bridges are
present between protein molecules.
Digestion has an important influence on the allergenicity of foods. An example of a group of enzymes
is the proteolytic enzymes. This group cleaves proteins into smaller peptides. Three examples of
enzymes belonging to this group are: pepsin, trypsin, and chymotrypsin. The enzymes are secreted
in different parts of the body and cleave different peptide bonds in proteins.
• Pepsin cleaves the c-terminal linkages of Leu, Phe, Trp or Tyr, unless preceded by Pro.
• Trypsin cleaves on the c-terminal side of Arg or Lys, unless followed by Pro.
You can assume that the C-terminal of the peptide is situated on the right side of the peptide. The
amino sequence is read from the left to the right.
If you want to know what type of proteins are present, you should measure the protein
composition. The protein content is the total amount of protein that is present in your sample. You
can calculate this from the data of the protein composition by adding up all the values of the protein
The LOD and LOQ respectively stand for Limit of Detection and Limit of Quantification
The LOD should be lower than the concentration required in a food to cause an adverse response. .
The LOD can be outside the linear range of the method, therefore also the LOQ is important.
The MLA stands for maximal level allowed in the product.
The MLA should be higher than the LOD because it should be possible to make a deliberate choice to
reject or accept the product.
An allergen loses its allergenicity if the epitopes are separated. The molecules with epitopes further
apart have a higher change of cleavage between the epitopes. Be aware that this is the chance, and
the result depends on the type of enzyme that is used. Each enzyme cleaves different bonds.
1. To dissolve it is easier to surround a smaller molecule by the molecules of the solvent.
2. A higher temperature results in a higher kinetic energy which is in favor of the solution
rate.
3. Hydrophobic will be soluble in a hydrophobic liquid; water is hydrophilic.
4. Crushing the matrix will increase the surface area. A larger surface area increases the
ease of extraction.
5. Tannins hinder the extraction rate of a molecule from the matrix, because the tannins
bind to the
6. The DTT reaches all parts of the protein faster if the solution is mixed
Bradford reagent (Coomassie Brilliant Blue) forms a colored complex with protein. The intensity of
the color is an indication for the concentration of protein. The process itself is fast and easy. The
binding process is finished after approximately two minutes and has a proper stability for around
, one hour, which gives you enough time to analyze the samples with a spectrophotometer. For this
method, a calibration curve is necessary to conclude what the protein concentration is.
Bradford method:
As a result of this method, a color is formed.
1. This method does not identify the epitopes on a protein.
2. It is not possible to make a distinction between the different proteins.
3. Measuring the total amount in the product is not possible since turbidities are removed.
This could be insoluble proteins. Therefore, only soluble proteins are measured.
4. Indeed, the protein that is soluble will end up in the sample that you will measure.
If the color changes from red to blue, the wavelength of the light decreases. Blue has a lower wave
length than red. This means that when a blue color is visible, the other wavelengths are absorbed.
This results in an increase in absorbance maximum when the color changes from red to blue.
Bradford measures the amount of protein and is thus quantitative. Coomassie Brilliant Blue binds to
proteins and not specifically to epitopes. It is only possible to measure the amount of protein and
not the type of epitope. The measurement of the developed color is performed with light of 595 nm,
this is inside the visible region.
When to use the bradford method
1. To measure the protein content directly from a solid potato FALSE
2. To have a good indication of the amount of protein before SDS-PAGE TRUE
3. To measure the protein content of a transparent solution TRUE
4. To measure the protein content of a turbid solution FALSE
PCR testing
PCR polymerase chain reaction test detects specific DNA fragments by a repeated amplification
procedure with DNA polymerase in which temperature plays an important role. A primer binds to a
specific DNA sequence and acts as a starting point for the DNA copying. An amino acid sequence
codes for a protein. The protein might have epitopes that can cause an allergic reaction. However,
One of the disadvantages is that DNA is relatively heat labile.
Because DNA is heat labile, it is possible that you do not find all the specific DNA sequences. This is
an example of a false negative. For a correct conclusion it is important to include a control. This
shows you if the method works and if the DNA degrades during the method.
The primer binds to single strand DNA in this method. Finally, this method can result in false positives
because it is an indirect method. DNA codes for protein but do not indicate if the protein is present.
1. in the last question you learned that PCR detects DNA sequences. The sequence codes for
a protein, which does not imply that the protein is also present.
2. Pcr is a qualitative method because the amplification procedure makes it impossible to
quantify.
3. the specific temperatures and media that are needed for this technique make it difficult
to perform at home.
4. in the laboratory this technique is easy to perform and even a small amount of material is
already enough for the amplification procedure.
5. in this method you are looking for the presence of a specific DNA sequence.
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