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Principle:
The PCR technique is based on the enzymatic replication of DNA. In PCR, a short segment
of DNA is amplified using primer mediated enzymes. DNA Polymerase synthesises new
strands of DNA complementary to the template DNA. The DNA polymerase can add a
nucleotide to the pre-existing 3'-OH group only.
Procedure:
Add forward and reverse primer- taq polymerase- dNTP- autoclaved water/nuclease free
water- DNA sample (cDNA)
Denaturation at 94 for 20 to 30 sec- annealing ranging from 55-65 for 30-40sec- elongation
94 for 30sec to 1min for 30-35 cycle
Application: I used it to amplify DNA molecules to get the better yield
SDS-PAGE:
Principle:
The principle of SDS-PAGE is based on separating proteins primarily according to their
molecular weight. It utilizes the denaturing detergent SDS to ensure that proteins are
uniformly charged and eliminates the influence of their native structure or charge differences
during electrophoresis.
Procedure:
Generally, we make 10%, 12%, and 15% gel. Different companies use different
measurements for components, so I am not mentioning the proportions. First we make
resolving gel. For that after fixing the gel casting apparatus properly we mix acryl mix, 10%
SDS, 8.4 pH Tris HCl buffer, and 10% APS and temed very quikly in a small vial then pour it
into the apparatus and immediately add 100% isopropanol to remove the bubbles. Then wait
for about 25 mins. After 25mins remove the isopropanol. Add now its time for making
stacking gel. Every other components will be same but in different amount obviously. Only
the pH for 10% SDS will be 6.8. after mixing all the components add it to the gel casting
apparatus and carefully place the comb and wait for 25mins.
In the mean time we can make the protein samples. And we have to heat the samples for 3min
at 95c.
We set the gel apparatus. Place the gel plates. Add running buffer. And carelly remove the
comb. Then we add samples and protein ladder. And we run the gel at about 130mV for about
45min – 1hr which varies according to the size of the protein molecule.
Application:
SDS-PAGE can determine the size and purity of proteins by separating them based on
their molecular weight. Post-translational modifications: SDS-PAGE can analyze
post-translational modifications to proteins.
Protein-protein interactions: SDS-PAGE can determine protein-protein
interactions.
and for western blot it it necessary to separate proteins in sds page before blotting
procedure
Western blot:
The principle of Western blotting (WB) is the use of antibodies to detect and identify proteins
by separating them based on their molecular weight and electrical properties.
Procedure:
Make a sandwich of 2 filter paper, 1 nitrocellulose membrane, gel, and again 2 filter paper.
Before making this sandwich filter papers and nitrocellulose membrane should be soaked in
transfer buffer. Nitrocellulose membrane should be in the anode face and gel should be in the
cathode face so that the proteins can be transferred from cathode to anode. Run the machine
for 30 min to 45min at 120mv, it can vary.
Next step is blocking and antibody incubation. For that the membrane should be taken in a
box and incubate it with blocking buffer for 1 hr with gentle rocking
Then add primary antibody and incubate it overnight at 4 c
Next day store again the primary antibody and wash the membrane 3 times with wash buffer
Then add secondary antibody and incubate it 3hrs in room temperature
Discard the antibody and wash the membrane 3 to 5times with wash buffer
After that the chemilumicient substrate should be added in the membrane which will cause
light to be emitted where HRP conjugated antibodies are present. Then the membrane can be
visulalized under chemi doc for the detection of desired protein band
Application:
Western blotting is a technique used in scientific and clinical research to detect and analyze
proteins
Western blotting is used to evaluate protein expression levels in cells and to purify proteins.
Western blotting can determine the extent of post-translational modifications and verify
protein expression in cloning applications.
Western blotting can identify proteins in a mixture by separating them based on their
molecular weight.
ELISA:
Principle:
ELISA works on the principle that specific antibodies bind the target antigen and detect the
presence and quantity of antigens binding. In order to increase the sensitivity and precision of
the assay, the plate must be coated with antibodies with high affinity. ELISA can provide a
useful measurement of antigen-antibody concentration.
Procedure:
An antibody is attached to a polystyrene plate which is a solid surface and is attracted
or has an affinity towards bacteria, other antibodies and hormones.
A microtiter coated with antigen is filled with this antigen-antibody mixture after
which free antibodies are removed by washing.
A second antibody specific to primary antibody is added which is usually conjugated
with an enzyme.
Free enzyme-linked secondary antibodies are removed by washing the plate.
Finally, the substrate is added. The substrate is converted by the enzyme to form a
coloured product, which can be measured by spectrophotometry.
The Enzyme Linked Immunosorbent Assay (ELISA) utilizes an enzyme with its substrate to
detect the antigen-antibody reaction in a positive test. The Immunofluorescence Assay ( IFA)
utilizes a fluorochrome-tagged antibody to detect a positive test. The assay may be direct or
indirect.
To detect an unknown protein using ELISA, you would first need to generate specific
antibodies against the protein, then use a "sandwich ELISA" method where you coat the plate
with a capture antibody that binds to a known epitope on your target protein, add your
unknown protein sample, and finally use a detection antibody (also specific to the target
protein) linked to an enzyme to visualize the bound protein by adding a substrate and
measuring the color change produced; the intensity of the color change will correspond to the
amount of the target protein present in your sample.
Generate antibodies:
Purify the unknown protein (if possible) to generate antibodies against it by
injecting it into an animal (e.g., mouse, rabbit).
Collect the antibodies from the animal's serum and purify them to obtain
specific antibodies against your target protein.
IFA:
Principle:
Immunofluorescence is a type of assay performed on biological samples to detect
specific antigens in any biological specimen or sample and vice-versa. The specificity of
antibodies to their antigen is the base for immunofluorescence.
Procedure:
Direct- Single antibody i.e. primary antibody is used that is chemically linked to
a fluorochrome. If the antigen is present, the primary antibody directly reacts with it and
fluorescence can be observed under the fluorescent microscope.
Indirect- Double antibodies are used i.e. primary and secondary antibodies. The primary
antibody is not labeled and a fluorochrome-labeled secondary antibody is used for detection.
The antigen used is known and it binds to the specific primary antibodies of interest in the
sample. The secondary antibody then binds to the Fc region of the primary antibody.
1. Fixing of a known antigen on a slide.
2. The specimen to be tested is applied to the slide. (taken from human body)
3. Incubation and careful washing with PBS.
4. A secondary antibody (e.g., fluorescently labeled anti-IgG) is added.
5. Incubation and careful washing again with PBS.
6. Observed under the fluorescence microscope.
To detect an unknown protein using immunofluorescence assay, you would need to first
generate a specific antibody against that protein, then use that antibody in an
immunofluorescence staining procedure to visualize the protein's location within cells or
tissues under a fluorescent microscope; this process typically involves generating a primary
antibody against the unknown protein and then using a secondary antibody tagged with a
fluorophore to detect the primary antibody binding site.
Key steps involved:
Protein purification and antibody generation:
Isolate the unknown protein: Purify the unknown protein from a sample
(e.g., cell lysate) using techniques like chromatography or electrophoresis to
obtain a concentrated protein sample.
Immunization: Inject the purified protein into an animal (e.g., mouse, rabbit)
to stimulate the production of antibodies against it.
Antibody collection: Collect serum from the immunized animal and purify
the specific antibodies against the unknown protein using affinity
chromatography.
Immunofluorescence staining procedure:
Sample preparation: Prepare cells or tissue sections by fixing them on a
microscope slide to preserve cellular morphology.
Permeabilization: If the unknown protein is intracellular, permeabilize the
cells using a detergent to allow antibodies to access the protein.
Blocking: Incubate the sample with a blocking solution to prevent non-
specific antibody binding.
Primary antibody incubation: Apply the primary antibody (specific to the
unknown protein) to the sample and incubate to allow binding.
Washing: Rinse the sample to remove unbound primary antibody.
Secondary antibody incubation: Apply a secondary antibody conjugated to a
fluorophore (e.g., Alexa Fluor) that recognizes the primary antibody species
and incubate.
Washing and mounting: Wash the sample again and mount with an
appropriate mounting medium.
Visualization:
Fluorescence microscopy: Examine the sample under a fluorescence
microscope to visualize the fluorescent signal indicating the location of the
unknown protein within the cells or tissue.
Genetic transformation:
The genetic transformation principle is the process by which an organism takes up DNA from
its environment and incorporates it into its own genome. This process is also known as
transformation.
Bacterial cloning inside a plasmid involves introducing a gene or DNA fragment into a
plasmid vector, which is then introduced into bacteria (usually E. coli) for replication and
possibly protein expression.
Chromatography:
Cell culture:
Considerations for creating an optimized cell culture environment for your cells:
pH levels Most normal mammalian cell lines grow well at pH 7.4, and there is very little
variability among different cell strains. However, some transformed cell lines have been
shown to grow better at slightly more acidic environments (pH 7.0 – 7.4), and some normal
fibroblast cell lines prefer slightly more basic environments (pH 7.4 – 7.7). Insect cell lines
such as Sf9 and Sf21 grow optimally at pH 6.2.
CO2 levels The growth medium controls the pH of the culture and buffers the cells in culture
against changes in the pH. Usually, this buffering is achieved by including an organic (e.g.,
HEPES) or CO2-bicarbonate based buffer. Because the pH of the medium is dependent on
the delicate balance of dissolved carbon dioxide (CO2) and bicarbonate (HCO3–), changes in
the atmospheric CO2 can alter the pH of the medium. Therefore, it is necessary to use
exogenous CO2 when using media buffered with a CO2-bicarbonate based buffer, especially
if the cells are cultured in open dishes or transformed cell lines are cultured at high
concentrations. While most researchers usually use 5 – 7% CO2 in air, 4 – 10% CO2 is
common for most cell culture experiments. However, each medium has a recommended CO2
tension and bicarbonate concentration to achieve the correct pH and osmolality. Optimal
temperatures for various cell lines: The optimal temperature for cell culture largely depends
on the body temperature of the host from which the cells were isolated, and to a lesser degree
on the anatomical variation in temperature (e.g., temperature of the skin may be lower than
the temperature of skeletal muscle). Overheating is a more serious problem than underheating
for cell cultures; therefore, often the temperature in the incubator is set slightly lower than the
optimal temperature.
Most human and mammalian cell lines are maintained at 36°C to 37°C for optimal growth. ●
Insect cells are cultured at 27°C for optimal growth; they grow more slowly at lower
temperatures and at temperatures between 27°C and 30°C.
There are two basic types of incubators, dry incubators and humid CO 2 incubators. Dry
incubators are more economical, but require the cell cultures to be incubated in sealed flasks
to prevent evaporation. Placing a water dish in a dry incubator can provide some humidity,
but they do not allow precise control of atmospheric conditions in the incubator. Humid CO2
incubators are more expensive, but allow superior control of culture conditions. They can be
used to incubate cells cultured in Petri dishes or multiwell plates, which require a controlled
atmosphere of high humidity and increased CO 2 tension.
Serum: Immediate survival (a balanced salt solution with specified pH and adequate osmotic
pressure) Prolonged survival (a balanced salt solution in addition with serum, or appropriate
formulation of organic compounds. Indefinite growth Specialized functions Artificial media
may be classified into following types: Serum containing media Serum-free media Protein
free media Chemically defined media Serum is the yellowish liquid and is a transparent
content that remains left over after the removal of fibrin and cells from the blood. 11 2-10%
of serum is often contained by normal growth media. The most commonly employed
supplement in animal cell culture is fetal bovine serum (FBS). It supplies the basic nutrients
for cells. It also contains several hormones and various growth factor. In addition to it, it also
acts as a buffer.
Serum containing media: In animal cell culture media, fetal bovine serum is the most
common supplement. In order to supply an optimal culture medium, it is employed as an
economical supplement. Serum supplies carriers water-insoluble nutrients, protease
inhibitors, hormones and growth factors and binds and neutralizes the toxic moieties. Serum
free media: In case of immunological studies, presence of serum in media can result to
serious misinterpretations. In general, these media are specifically designed to promote the
culture of a single type of cell and incorporate specified amounts of purified growth factors,
lipoproteins and other proteins normally supplied by the serum.
A. Primary cell culture This is the cell culture obtained straight from the cells of a host tissue.
The cells dissociated from the parental tissue are grown on a suitable container and the
culture thus obtained is called primary cell culture. Such culture comprises mostly
heterogeneous cells and most of the cells divide only for a limited time. However, these cells
are much similar to their parents. Depending on their origin, primary cells grow either as an
adherent monolayer or in a suspension.
Adherent cells These cells are anchorage dependent and propagate as a monolayer. These
cells need to be attached to a solid or semi-solid substrate for proliferation. These adhere to
the culture vessel with the use of an extracellular matrix which is generally derived from
tissues of organs that are immobile and embedded in a network of connective tissue.
Fibroblasts and epithelial cells are of such types. When the bottom of the culture vessel is
covered with a continuous layer of cells, usually one cell in thickness, these are known as
monolayer cultures. Majority of continuous cell lines grow as monolayers. As being single
layers, such cells can be transferred directly to a cover slip to examine under a microscope.
Suspension cells Suspension cells do not attach to the surface of the culture vessels. These
cells are also called anchorage independent or non-adherent cells which can be grown
floating in the culture medium. Hematopoietic stem cells (derived from blood, spleen and
bone marrow) and tumor cells can be grown in suspension. These cells grow much faster
which do not require the frequent replacement of the medium and can be easily maintained.
These are of homogeneous types and enzyme treatment is not required for the dissociation of
cells; similarly these cultures have short lag period.
Confluent culture and the necessity of sub-culture After the cells are isolated from the tissue
and proliferated under the appropriate conditions, they occupy all of the available substrate
i.e. reach confluence. For a few days, it can become too crowded for their container and this
can be detrimental to their growth, generally leading to cell death if left for a long time. The
cells thus have to be subculture i.e. a portion of cells is transferred to a new vessel with fresh
growth medium which provides more space and nutrients for the continual growth of both
portions of cells. Hence subculture keeps cells healthy and in a growing state. A passage
number refers specifically to how many times a cell line has been sub-cultured. In contrast
with the population doubling level in that the specific number of cells involved is not
relevant. It simply gives a general indication of how old the cells may be for various assays.
B. Secondary cell culture and cell line When a primary culture is sub-cultured, it is known as
secondary culture or cell line or sub clone. The process involves removing the growth media
and disassociating the adhered cells (usually enzymatically). Sub-culturing of primary cells to
different divisions leads to the generation of cell lines. During the passage, cells with the
highest growth capacity predominate, resulting in a degree of genotypic and phenotypic
uniformity in the population. However, as they are sub-cultured serially, they become
different from the original cell. On the basis of the life span of culture, the cell lines are
categorized into two types:
Finite cell lines The cell lines which go through a limited number of cell division having a
limited life span are known as finite cell lines. The cells passage several times and then lose
their ability to proliferate, which is a
genetically determined event known
as senescence. Cell lines derived from
primary cultures of normal cells are finite
cell lines.
Continuous cell lines When a finite cell line undergoes transformation and acquires the ability
to divide indefinitely, it becomes a continuous cell line. Such transformation or mutation can
occur spontaneously or can be chemically or virally induced or from the establishment of cell
cultures from malignant tissue. Cell cultures prepared in this way can be sub-cultured and
grown indefinitely as permanent cell lines and are immortal. These cells are less adherent,
fast growing, less fastidious in their nutritional requirements, able to grow up to higher cell
density and different in phenotypes from the original tissue. Such cells grow more in
suspension. They also have a tendency to grow on top of each other in multilayers on culture-
vessel surfaces.
Media preparation:
Parasite: RPMI- Roswell Park Memorial Institute Medium
Here are some steps for preparing RPMI 1640 media:
1. Measure out 90% of the final volume of water at a temperature of 15–20°C.
2. Use deionized, pyrogen-free, or bidistilled water.
3. If using a powder, add the entire contents of the package immediately after opening.
4. When the powder is completely dissolved, add sodium hydrogen carbonate
(NaHCO3) at a rate of 0.850 g per liter of the final medium.
5. Add supplements before filtration or aseptically to sterile medium.
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