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We have to measure the level of specific protein within a
biological sample using rat cardiovascular tissue. A powerful tool called
Western Blotting is used for this purpose.

Abstract:

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Western blotting is a significant technique used in a cell
and molecular biology to ascertain the levels of specific proteins. Researchers
are able to identify specific proteins from a complex mixture of proteins
extracted from cells within a biological sample using this Western blot
technique. The technique uses three elements to accomplish this task:

•             Transfer
to a solid support

•             Separation
by size

•             Marking
target protein; using a proper primary and secondary antibody to visualize

This lab report based on research related to western blot
and measuring specific protein will attempt to explain the technique and theory
behind western blot, and offer some ways to troubleshoot. We are using rat
cardiovascular tissue for this technique. Using Western blotting technique, we
are going to measure the levels of Akt.

We are using SDS-PAGE to separate proteins on the basis of
their size. The SDS gives proteins an overall negative charge, which allows
them to migrate towards the anode (+) during the process of electrophoresis. At
the end, we are going to use Immunodetection to specifically identify Phospho-Akt.
For this, the membrane will be probed with a primary antibody, raised against
Phospho-Akt protein.

Introduction:

Clinical Background of Western blotting technique:

We are going to use western blotting technique to measure
specific protein in a biological sample. We are using rat cardiovascular
tissues as our biological sample. There are evidences that following
reperfusion, some specific survival pathways are activated in order to salvage
cardiac myocytes which have been affected by the ischemic injury. These
pathways are known as the Reperfusion Injury Salvage Kinase (RISK) pathways
(Davidson et al., 2006). Ipratropium is a medicine used in this western
blotting technique. Ipratropium bromide is sold under the trade name of Atrovent among
some others. It is a medication which is used for opening up the medium
and large airways in human lungs. It is basically used to treat the
symptoms of asthma and chronic obstructive pulmonary disease. It is
used by a nebulizer as well as an inhaler too. Ipratropium was
developed in Germany in 1976 and was approved in USA for the use first time in
1986. The clinical background of western blotting technique tells us that this technique
has been used for the past three or four decades widely in laboratories. Yet,
there has been comparatively little discussion of how the advancement in
Western blotting technique is going to affect its clinical use in future. But
the fact is that Western blotting technique is still used many times one way or
another, to diagnose some of the major diseases including hepatitis C also called HCV, HIV,
Lyme disease, and syphilis.

Technique:

Cell lysis is an important part of Western blotting technique
in which proteins are extracted from biological samples. Specific proteins can
be ascertained from any kind of biological sample, such as a cell or a tissue.
For our lab sessions we will be using the rat cardiovascular tissues as our biological
samples to perform Western blotting experiment. One of the major proteins that
is activated in these signaling cascades is known as AKT (other name used for
it is Protein Kinase B). Specific survival pathways are activated in order to
salvage cardiac myocyte.

·        
Cell lysis

·        
Preparing the gel

·        
Electrophoresis

·        
Electro transfer

·        
Blocking and antibody incubation

1. Using cell lysis technique we are going to extract protein
from adherent cells by washing the cells in the flask known as tissue culture
flask. Then we will add cold PBS and after rocking gently, we will discard PBS.
Add PBS and by using cell scraper, you can easily dislodge the cells. After
some precise calculations, timings and following some important steps that we
will discuss in detail in discussion below, we will be then able to measure the
concentration of protein using a
spectrophotometer.
2. Gel preparation is another significant part of extracting protein from
tissue or cell. Solution of stacking gel is prepared of 10% quantity. Holding the
glass plates, add stacking gel solution carefully until the level equals
the level of green bar. Add H2O (water) to the top of this gel. Wait for almost
15–30 minutes. Wait until the gel starts turning solidified. It will be easier
to add gel to the glass using a suction pipette.
3. Electrophoresis method is used and then pours the running buffer into the electrophorator. Place gel inside the
electrophorator and connect to a power supply.  Make sure that the buffer is covering the
gel completely. After this, run the gel with higher voltage at 140 V for
stacking the gel. You can lower the voltage at almost 60V for separation of gel.
Run gel for up to 60 minutes.
4. Electro transfer will be used by creating a sandwich like
Sponge, three filter papers, Gel PVDF, three filter papers respectively. 4
degree should be maintained and the sandwich should be relocated to the transfer
apparatus. Ensure that the sandwich is covered with the buffer and meanwhile add
transfer buffer to the apparatus.
5. Block the membrane with almost 5-7% skim milk inside TBST* for 60
minutes. A shaker is used to incubate the membrane with additional help
of antibody. Do wash the membrane at least 3 times with TBST for 10
minutes. Prepare ECL mix. Incubate the membrane for almost 120 seconds. See the
result in the room with dim lights. 

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