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    Western%20Blotting(safia)%20(1)

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    Nazwa anha
    The lab report details Experiment 09, which utilized western blotting to detect specific target proteins from Lactobacillus based on their molecular weight. The experiment involved preparing protein samples, transferring them to a membrane, and using antibodies for detection, resulting in two protein bands at approximately 68 kDa and 40 kDa. The report also discusses the methodology, results, and necessary precautions to minimize contamination during the experiment.

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    Western%20Blotting(safia)%20(1)

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    Nazwa anha
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    The lab report details Experiment 09, which utilized western blotting to detect specific target proteins from Lactobacillus based on their molecular weight. The experiment involved preparing protein samples, transferring them to a membrane, and using antibodies for detection, resulting in two protein bands at approximately 68 kDa and 40 kDa. The report also discusses the methodology, results, and necessary precautions to minimize contamination during the experiment.

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    The lab report details Experiment 09, which utilized western blotting to detect specific target proteins from Lactobacillus based on their molecular weight. The experiment involved preparing protein samples, transferring them to a membrane, and using antibodies for detection, resulting in two protein bands at approximately 68 kDa and 40 kDa. The report also discusses the methodology, results, and necessary precautions to minimize contamination during the experiment.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    0 views5 pages

    Western%20Blotting(safia)%20(1)

    Uploaded by

    Nazwa anha
    The lab report details Experiment 09, which utilized western blotting to detect specific target proteins from Lactobacillus based on their molecular weight. The experiment involved preparing protein samples, transferring them to a membrane, and using antibodies for detection, resulting in two protein bands at approximately 68 kDa and 40 kDa. The report also discusses the methodology, results, and necessary precautions to minimize contamination during the experiment.

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    BRAC University

    Department of Mathematics and Natural Sciences

    Biotechnology Program

    BTE355

    Biotech Lab IV

    Section 05

    Lab Report

    Experiment 09

    Submitted By

    Safia Maherzia

    ID : 22336025

    Fall 2024
    Experiment Number: 09

    Experiment Title: Western Blotting

    Objective:

    The purpose of this experiment was to use western blotting to detect a specific target protein
    from Lactobacillus, relying on its previously established molecular weight.

    Principle:

    Western blotting is a laboratory technique employed to identify specific protein molecules from
    within a complex protein mixture, such as the total protein content of a cell or tissue. This
    technique can also be used to determine a protein's size and measure its level of expression. The
    name "western blot" is derived from its similarity to the earlier-developed Southern blot
    technique.

    The first step in a western blot involves preparing the protein sample by mixing it with the
    detergent sodium dodecyl sulfate (SDS). SDS causes the proteins to unfold into linear chains and
    coats them with a negative charge. The proteins are then separated according to their molecular
    weight using SDS-PAGE. After separation, the proteins are transferred from the polyacrylamide
    gel to a blotting membrane. Common membrane materials include nitrocellulose and
    polyvinylidene fluoride (PVDF). Nitrocellulose is typically used for single applications,
    especially with smaller proteins, whereas PVDF is more suitable for multiple uses, particularly
    with larger proteins. Following the transfer, the membrane now holds all the protein bands
    initially present in the gel.

    The next step is blocking, which prevents non-specific binding.

    Bovine serum albumin (BSA) is commonly used as the blocking agent, binding to all
    unoccupied sites on the membrane, effectively preventing the primary antibody from binding
    directly to the membrane. The membrane is then incubated with primary antibodies that
    specifically bind to the target protein. After this incubation, any unbound primary antibody is
    washed away, and the membrane is incubated with a secondary antibody. This secondary
    antibody specifically recognizes and binds to the primary antibody. Importantly, the secondary
    antibody is linked to a reporter enzyme that generates a detectable signal, either a colored
    product or light emission, enabling easy detection and imaging of the target protein. This series
    of steps allows for the specific detection of a single protein within a complex mixture.

    Reagents:

    1
    ● Transfer buffer (Towbin buffer)
    Apparatus:
    ● Whatman filter paper
    ● Nitrocellulose membrane
    ● Foam pads
    ● Gel containing protein sample
    ● Blotting cassette
    ● Blotting machine and power supply

    Methods:

    Preparation for Gel Sandwich Assembly:

    ●​ The destained gel from the SDS-PAGE procedure was obtained.


    ●​ The transfer buffer was cooled overnight at 4°C.
    ●​ The gel was then pre-equilibrated in the chilled transfer buffer for 15 minutes.
    ●​ Blotting paper (Whatman filter paper) and the membrane were cut to the same
    dimensions as the gel and immersed in the transfer buffer for 15 minutes.
    ●​ Two foam pads were also soaked in the transfer buffer for 15 minutes.

    Assembling the Gel Sandwich:

    ●​ The black side of the blotting cassette was placed downwards on a flat surface.
    ●​ A pre-soaked foam pad was placed inside the black half of the blotting cassette, followed
    by three layers of pre-soaked filter paper.
    ●​ The gel was then positioned on top of the prepared filter paper layers.
    ●​ A glass rod, moistened with a transfer buffer, was carefully rolled over the gel surface to
    eliminate any trapped air bubbles.
    ●​ The transfer membrane was carefully aligned and placed directly onto the gel.
    ●​ Three additional layers of pre-soaked filter paper were placed on top of the membrane.
    ●​ A pre-soaked foam pad was placed on top to complete the sandwich assembly.
    ●​ The assembled blotting cassette was then inserted into the blotting module.

    Setting up the Buffer Tank:

    ●​ The buffer tank was filled with a transfer buffer to approximately half or two-thirds of its
    capacity.
    ●​ The cooling system was connected to the designated connectors at the base of the tank.
    ●​ The blotting module was carefully slid into the buffer tank.
    ●​ More transfer buffers were added to the tank until it reached the indicated fill line.

    2
    ●​ The Bigfoot Safety Lid was placed on the buffer tank, and the power cords were
    connected.
    ●​ The transfer was carried out at a constant voltage of 25 V for 40 minutes.

    Figure 9.1: The result of Western blotting

    Results and Observations:

    Two protein bands were visualized, with estimated molecular weights of about 68 kDa and 40
    kDa.

    Discussion:

    The goal of this experiment was to verify the presence of specific proteins previously isolated
    from Lactobacillus. The molecular weights of the observed proteins were determined by
    comparing them to a standard protein ladder. This comparison revealed two distinct bands,
    corresponding to approximate molecular weights of 68 kDa and 40 kDa.

    A prominent, sharp band at 68 kDa, visible in multiple lanes, confirmed the consistent presence
    of a protein of this size. The greater intensity of this band suggested a higher concentration of
    this protein. Faint bands were observed at 40 kDa, indicating a lower level of expression for a

    3
    protein of this molecular weight. Consequently, the presence of bands at the expected molecular
    weights validated the detection of the targeted proteins.

    Because the experiment was performed in a shared BSL-1 laboratory without a laminar flow
    hood, the risk of contamination from other researchers in the same space was elevated. To
    minimize contamination and ensure safety, gloves were worn during all handling of equipment
    and the gel. The membrane was oriented with its shiny side facing upwards during assembly of
    the blotting cassette. Freshly prepared buffer was used to maintain the correct pH. The
    membrane was cut to the same size as the foam pads to ensure efficient assembly of the blotting
    cassette.

    The polyacrylamide gel, containing the neurotoxic substances acrylamide and bis-acrylamide,
    was handled with caution. Due to its thinness, the gel was fragile and could easily tear if not
    handled carefully. The gel was stored immersed in a staining solution to prevent it from drying
    out. All materials used in the blotting process, including the foam pads, membranes, and filter
    paper, were pre-soaked in a buffer to facilitate ion movement during the transfer.

    Precautions:

    1.​ The work area should have been sterilized both before and after the experiment.
    2.​ The membrane, blotting paper, and foam pads should have been soaked in buffer solution
    prior to use.
    3.​ The gel should have been handled carefully to prevent tearing.
    4.​ The appropriate electrophoresis run time and voltage should have been maintained.
    5.​ Gloves should have been worn during all handling of reagents and the gel.
    6.​ The gel should have been kept immersed in the staining solution.

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