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Determination of isoelectric point
Determination of isoelectric point












determination of isoelectric point

The pipette was then quickly blown out each time as much of the casein enters the test tube each time. Fourth, 1ml of casein 0.5g/1 in 0.1M sodium acetate was pipetted into each test tube.Third, the designated amount of 1.0 acetic acid according to table 1 was added to test tube 9.Second, the designated amounts of 1.0 M acetic acid according to table 1 was added to test tubes 3,4,5,6,7 and 8.If the process was carries out the other way round, the higher concentration of the 1.0 M acid would raise the acidity level of the 0/01 acid. In order to reduce the chances of contamination, the designated amounts of 0.01M acetic acid was pipetted first to test tubes 1 and 2 because the 0.01 M acetic acid is the least concentrated acid of the 3 acids.

determination of isoelectric point

The acetic acid was then pipetted into each test tube according to the values in the table 1.

  • Following the designated volumes required on table 1, the volumes of distilled water was then pipetted into each test tube.
  • This is important because all solution are a similar colour.
  • In order to distinguish between the different acidity levels contained in each test tube, the 9 test tubes were labelled from 1-9.
  • The calorimeter is switched ON to allow it to “warm up”.
  • The object of this experiment is to determine the isoelectric point of casein (protein), which can be precipitated from the solution. A globular protein such as a casein becomes increasingly insoluble as it approaches its isoelectric point. The ph at which the protein is electrically neutral is known as the isoelectric point. Globular proteins are hydrophobic proteins which in certain external condition are soluble in eater. The paper also shows that using this technique it is possible to estimate the pI values for a wide range of proteins measuring at only two pH values, suggesting that this technique is rapid and accurate on small volume samples.įigure 1: shows a diagram of the microfluidic chip used to determine the pI of the target proteins without a spatial pH gradient and instead using a temporal gradient.Determination of isoelectric point of protein (casein).Ĭasein is a globular colloidal protein. The technique requires low voltages and low sample consumption. The paper shows that this method is successful in determining the pI using this new technique without the requirement of generating and maintaining pH gradients which is often challenging for other techniques. To demonstrate the effectiveness of this method the pI of 7 different proteins of known pI were tested β-lactoglobulin, ribonuclease A, ovalbumin, human transferrin, ubiquitin and myoglobin. The approach exploits temporal rather than spatial pH gradients. using a microfluidic system built in house design a new technique to determine a protein’s isoelectric point (pI) based on microfluidic free-flow electrophoresis (μFFE).

    #Determination of isoelectric point free

    The ability to conduct measurements in free solution thus provides the basis for the rapid determination of isoelectric points of proteins under a wide variety of solution conditions and in small volumes. To demonstrate the general approachability of this platform, they have measured the isoelectric points of representative set of seven proteins, bovine serum albumin, β-lactoglobulin, ribonuclease A, ovalbumin, human transferrin, ubiquitin and myoglobin in microlitre sample volumes. In particular, in this approach, the pH of the electrolyte solution is modulated in time rather than in space, as in the case for conventional determinations of the isoelectric point. Here, Łapińska et al., introduce a gradient-free approach, exploiting a microfluidic platform which allows us to perform rapid pH change on chip and probe the electrophoretic mobility of species in a controlled field. The majority of conventional methods for the determination of the isoelectric point of a molecule rely on the use of spatial gradients in pH, although significant practical challenges are associated with such techniques, notably the difficulty in generating a stable and well controlled pH gradient. The isoelectric point (pI) of a protein is a key characteristic that influences its overall electrostatic behavior.














    Determination of isoelectric point