Electrophoresis
Electrophoresis is a method of separating proteins based on their physical properties.
Proteins are separated upon;
 Net charge
 Size
 Shape
Principle:
The iso-electric point of various kinds of proteins are generally lower than 7.0, in
alkaline buffer solution (Ph 8.6), proteins are negatively charged, they will migrate
towards anode in electric field.
Procedure:
1) Immerse the membrane in the buffer 20mins before the experiment, take it out
and absorb with filter paper.
2) Add 3-5 microliter of sample on the rough side / well near the cathode, after the
sample has permeated into the membrane put the membrane in the electrophoresis
tank, coverslip is place to avoid evaporation and leave for 5 minutes.
3) Electric field is applied at room temperature, voltage 80v for 10 mins then increase
upto 100v for 30-45 minutes.
4) Immediately transfer the cellulose acetate to Ponceau 3R and fix and stain for 5
min.
5) Remove excess stain by washing for 5 min in the first acetic acid reservoir and
dry.
6) Densitometry and spectrophotometry can be used to identify the proteins.
Protease activity Assay
Sigma non-specific protease assay:
In the lab, it is often necessary to measure and/or compare the activity of proteases.
Sigma's non-specific protease activity assay may be used as a standardized
procedure to determine the activity of proteases, which is what we do during our
quality control procedures. In this assay, casein acts as a substrate. When the
protease we are testing digests casein, the amino acid tyrosine is liberated along with
other amino acids and peptide fragments. Folin & Ciocalteus Phenol, or Folin’s
reagent primarily reacts with free tyrosine to produce a blue colored chromophore,
which is quantifiable and measured as an absorbance value on the
spectrophotometer. The more tyrosine that is released from casein, the more the
chromophores are generated and the stronger the activity of the protease.
Absorbance values generated by the activity of the protease are compared to a
standard curve, which is generated by reacting known quantities of tyrosine with the
F-C reagent to correlate changes in absorbance with the amount of tyrosine in
micromoles. From the standard curve the activity of protease samples can be
determined in terms of Units, which is the amount in micromoles of tyrosine
equivalents released from casein per minute.
Micro Kjeldahl Method:
The Kjeldahl method is a means of determining the nitrogen content of organic and
inorganic substances. Although the technique and apparatus have been altered
considerably over the past 100 years, the basic principles introduced by Johan
Kjeldahl endure today.
The Kjeldahl method may be broken down into three main steps:
 Digestion
 Distillation
 Titration
1) Digestion:
The decomposition of nitrogen in organic samples utilizing a concentrated acid
solution. This is accomplished by boiling a homogeneous sample in concentrated
sulfuric acid. The end result is an ammonium sulfate solution.
2) Distillation:
Adding excess base (NaOH) to the acid digestion mixture to convert NH4 + to NH3,
followed by boiling and condensation of the NH3 gas in a receiving solution.
3) Titration:
To quantify the amount of ammonia in the receiving solution using boric acid, and
methyl orange as indicator.
The amount of nitrogen in a sample can be calculated from the quantified amount of
ammonia ions in the receiving solution.
Ramon Flocculation Test
Antigenic strength and purity of diphtheria toxoid as well as content of toxoid (and
toxin) in a sample can be expressed in flocculation value (Lf units).
All vaccine manufacturers use the original Ramon version of the flocculation test to
express Lf units in toxoids. The test is used as in process control the production
process and also to confirm the antigenic purity of bulk toxoid.
Ramon flocculation assay is an immunological binding assay in which the
flocculation value (Lf) of a toxoid (or toxin) is determined by the number of units of
antitoxin which, when mixed with the sample produces an optimally flocculating
mixture. Visible flocculation is formed as a result formation of antigen-antibody
complexes. Because calibration of antitoxins in International Units will be assay
dependent, antitoxins used in the Ramon assay must be directly calibrated against
the International Reference Reagent (IRR) for flocculation test.
The concentration of antitoxin thus determined may be indicated in Lf-equivalents
per milliliter (Lf-eq/ml). By definitiona, 1 Lf is the quantity of toxoid (or toxin) that
flocculates in the shortest time with 1 Lf-eq of specific antitoxin.
Info: When a serum gives a reaction at the usual temperature with the standard toxin
in 2, 3, or more hours, when it should give it in 1 hour, we know that the amounts of
toxin and antitoxin are not proportionate. The test is repeated several times until the
reaction is obtained within the proper time. The Ramon test, when the amounts of
toxin and antitoxin are proportionate is, in the opinion of the author, one of the most
reliable we have.
Determination of Hb Content by Sahli’s Method
Also called as acid hematin method.
We need:
1) Sahli’s apparatus: which consist of two lateral standard tubes and one central bigraduated tube.
2) Sahli’s Pipette: Capacity 20 ul
3) Blood sample (whole blood): in anti-coagulant tube
4) 0.1 N HCl & Distilled water
Reaction: Hemoglobin + 0.1N HCl
Acid Hematin (brown colour)
Globin
The resulting brown color is matched with the standard brown glass color.
Procedure: Place 0.1 N HCl into the central bi-graduated tube till the mark 2 on
the gram scale. Draw the blood into the sahli’s pipette exactly upto the mark 20.
Wipe off excess blood adhering to the pipette. Rinse the pipette by drawing the
acid in the tube several times. Mix, let stand for 10 minutes. Add distilled water
(drop by drop with mixing), until it matches the color of the standard tubes. Note
the reading in the g/dL scale.
Normal H.b range = 12 – 18 g/Dl
Mechanism of De-sensitization:
In desensitization immunotherapy the aim is to induce or restore tolerance to the
allergen by reducing its tendency to induce IgE production. Patients are desensitized
through the administration of escalating doses of allergen that gradually decreases
the IgE-dominated response. The objective of immunotherapy is to direct the
immune response away from humoral immunity and toward cellular immunity,
thereby encouraging the body to produce less IgE antibodies and more CD4+ T
regulatory cells that secrete IL-10 and TGF-β, which skews the response away from
IgE production.
OIT (oral immunotherapy) also creates an increase in allergen-specific IgG4
antibodies and a decrease in allergen-specific IgE antibodies, as well as diminished
mast cells and basophils, two cell types that are large contributors to allergic
reaction.
Mantoux Test
Mantoux test = PPD (purified protein derivative of Mycobacterium Tuberculosis)
= Tuberculin Test
This test determines that you have tuberculosis or not. T.B is a serious infection,
usually of the lungs. This bacteria spreads when you breathe in the air exhaled by a
person infected with T.B. The bacteria can remain in-active in body for years.
When immune system becomes weakened, T.B can become active and produce
symptoms such as




Fever
Weight loss
Coughing
Night sweats etc.
When T.B infects your body, your body becomes extra sensitive to certain
elements of the bacteria, such as the purified protein derivative. A PPD checks
your body’s current sensitivity.
Procedure: A small dose of the PPD (0.5ml) is injected intra-dermally in the fore
layer of the arm. After 2-3 days, observation will be made. You get many types of
result as follows;
1) Negative result (normal):
No firm bump forms at the test site, or a bump forms that is smaller than 5mm. No
induration and redness,
2) Positive result (Abnormal):
Induration + redness and bump formation gives 3 types of results;
3) Rare results:
Fasle-Positive: Have taken B.C.G vaccine, T.B with other than mycobacterium tuberculosis (can be M.Avium
complex)
False-Negative: Taking immune suppressants, Have diseases like cancer etc.
______The END______
Gram Staining
Acid Fast Stain