Drosophila melanogaster Western Blotting: Unveiling the Protein Landscape

Sania Vijayakumar

ENG 393

Professor Justice

23 April 2024
Introduction

The fruit fly, Drosophila melanogaster, has established itself as a cornerstone organism in
biological research. With its short life cycle, well-defined genetics, and relatively simple
anatomy, Drosophila offers a powerful model system for understanding complex biological
processes. Western blotting is a fundamental technique that plays a crucial role in dissecting
these processes at the molecular level. By enabling the visualization and quantification of
specific proteins within a fly sample, Western blotting provides invaluable insights into protein
expression patterns and regulation in Drosophila.

This guide presents a streamlined approach to Western blot analysis specifically designed for
Drosophila melanogaster. While Western blotting can be a multifaceted technique with
numerous variables to consider, this protocol focuses on the core steps involved in identifying a
single protein of interest in your fly samples. By following these steps and adhering to best
practices, researchers can gain valuable information about protein expression and function
within their chosen Drosophila model.

Western blotting serves as a powerful tool for protein analysis, functioning as a detective kit for
the cellular proteome. Imagine a complex mixture of proteins within a fly cell, akin to a crowded
crime scene. Western blotting allows researchers to isolate and identify a specific protein
suspect from this heterogeneous population. This is achieved by separating fly proteins based
on their size and then employing highly specific molecules called antibodies to target and
visualize the protein of interest. The resulting image, termed a Western blot, provides a
snapshot of the protein's presence and relative abundance within the sample.

This guide outlines the essential steps involved in a Drosophila Western blot experiment. It is
important to acknowledge that for intricate experiments or troubleshooting purposes,
researchers may need to consult detailed protocols and optimize specific conditions. However,
this simplified approach equips beginners with the foundation to initiate their exploration of
protein expression patterns in Drosophila.

Materials Needed

● Drosophila Samples: Adult flies, embryos, dissected tissues (e.g., heads, eyes) or

cultured Drosophila cells depending on the specific research question.

● Homogenization Buffer: A simple buffer like Tris-buffered saline (TBS) supplemented

with protease inhibitors to preserve protein integrity during sample preparation.

● Dounce Homogenizer: A handheld tissue grinder suitable for homogenizing small

samples like fly tissues.

● Electrophoresis Apparatus and Supplies: Basic equipment and reagents for

preparing protein gels (around 8% acrylamide).

 ● Protein Standards: Pre-stained protein ladder encompassing a broad range of

molecular weights for protein size estimation.

● Transfer Membrane: Nitrocellulose or PVDF membrane for efficient protein transfer.

● Blocking Buffer: Non-fat dry milk or bovine serum albumin (BSA) in TBS with Tween-20

to prevent non-specific antibody binding.

● Primary Antibody: A commercially available antibody specifically recognizing your

target Drosophila protein. Select an antibody validated for Western blotting in

Drosophila.

● Secondary Antibody: An HRP-conjugated secondary antibody specific to the host

species of your primary antibody (e.g., anti-rabbit HRP for a rabbit primary antibody).

● Western Blot Detection Reagents: Chemiluminescent substrate for HRP detection.

● Imaging System: A camera to capture the protein bands on the blot.

Methods

Phase 1: Sample Preparation

1. Sample Collection: Collect your Drosophila samples based on your research question.

Flash freeze the samples in

liquid nitrogen or store them at -

80°C to minimize protein

degradation.

Caution: Use appropriate

personal protective equipment

(PPE) when handling liquid

Figure 1. Sample collection and homogenization setup for
nitrogen. Drosophila samples.
 2. Homogenization: Using a chilled Dounce homogenizer, disrupt a small amount of your

frozen sample in a pre-chilled homogenization buffer. The homogenization buffer

volume and time may require slight adjustments depending on the sample type. The

goal is to thoroughly lyse the cells and release their protein content.

Caution: Exercise caution while using the Dounce homogenizer to prevent accidental

injury.

3. Centrifugation: Separate the cellular debris (pellet) from the protein-containing

supernatant (liquid) by centrifuging the homogenate at high speed (around 12,000 rpm)

for a few minutes. Discard the pellet and retain the supernatant for the next step.

4. Protein Quantitation (Optional): For more precise protein loading, quantify the protein

concentration in your supernatant using a method like Bradford assay. This helps

determine the optimal amount of protein to load onto the gel for efficient detection.

Phase 2: Protein Separation

1. Gel Preparation: Prepare a polyacrylamide gel with an acrylamide percentage suitable

for separating fly proteins. Typically, 8% gels offer a good resolution for most Drosophila

proteins.

2. Sample Loading and Electrophoresis: Load

your fly protein samples and protein standards

into the designated wells of the gel. Include a

positive control lane containing a lysate from

Figure 2. Diagram of SDS-PAGE gel preparation

for protein separation.
 wild-type flies or a cell line known to express your target protein. Apply a voltage and run

the electrophoresis according to the gel composition and expected protein sizes.

Phase 3: Protein Transfer

1. Transfer Assembly: Following standard protocols, prepare the transfer apparatus by

soaking the gel, membrane, and filter paper in the transfer buffer. Assemble the transfer

sandwich (gel, membrane, filter

paper) and ensure there are no air

bubbles between the layers. Place the

assembly in the transfer tank and set

up the electric current according to

Figure 3. Illustration of Western Blot Set-Up
the manufacturer's instructions. Transfer the proteins from the gel onto a nitrocellulose or

PVDF membrane.

Phase 4: Antibody Detection

1. Blocking: Immerse the membrane in a blocking buffer (e.g., 5% BSA in TBS-T) to

prevent non-specific antibody binding. Incubate the membrane for 1 hour at room

temperature or overnight at 4°C with gentle shaking.

2. Primary Antibody Incubation: Dilute the primary antibody in antibody incubation buffer

(e.g., 1% BSA in TBS-T) according to the manufacturer's recommendations. Incubate

the membrane with the

Figure 4. Illustration of antibody incubation and detection process in

western blotting.
 primary antibody for 1-2 hours at room temperature or overnight at 4°C with gentle

shaking.

3. Washing: Wash the membrane with TBS-Tween (0.1% Tween-20 in TBS) three times

for 10 minutes each to remove unbound primary antibodies.

4. Secondary Antibody Incubation: Dilute the species-specific HRP-conjugated

secondary antibody in an antibody incubation buffer. Incubate the membrane with the

secondary antibody for 1 hour at room temperature with gentle shaking.

5. Detection: Wash the membrane with TBS-Tween as before to remove unbound

secondary antibodies. Develop the blot using a chemiluminescent substrate according to

the manufacturer's instructions. Capture the chemiluminescent signal using X-ray film or

a chemiluminescence imaging system.

Understanding the Essentials: A Glossary

● Antibody: Y-shaped protein molecules that bind specifically to antigens (in this case,

your target protein).

● Blocking Buffer: A solution used to prevent non-specific antibody binding to the

membrane.

● Chemiluminescence: A light-emitting reaction used to visualize protein bands on a

Western blot.

● Electrophoresis: A technique that separates molecules based on their size and charge

using an electric field.

● Homogenization: The process of breaking down tissues into a homogenous

suspension.

● Protein Quantitation: The process of measuring the total protein concentration in a

sample.

● Protease Inhibitor: A molecule that inhibits the activity of proteases, enzymes that

degrade proteins.

● Supernatant: The liquid portion remaining after centrifugation, which contains soluble

components like proteins.

● Western Blot: A technique used to detect and visualize specific proteins in a sample

separated by electrophoresis.
 References

Jay, T. (2021). An overview of the sample preparation and ELISA assay protocols. In

Biorxiv.org.

https://www.biorxiv.org/content/biorxiv/early/2021/04/22/2021.04.21.440847/F7.large.jpg

Pellenz, S. (2013, December 6). Western blotting (immunoblot): Gel electrophoresis for

proteins. Antibodies-Online.com.

https://www.antibodies-online.com/resources/17/1224/western-blotting-immunoblot-gel-

electrophoresis-for-proteins/

The Principle and Procedure of Western Blot. (2018). In Creative Proteomics Blog.

https://www.creative-proteomics.com/blog/index.php/the-principle-and-procedure-of-

western-blot/

Wangler, M. (2017). 2 Schematic of SDS-Page electrophoresis. In ResearchGate.

https://www.researchgate.net/figure/Schematic-of-SDS-Page-electrophoresis-

Polyacrylamide-two-part-gel-composed-of-a-stacking_fig21_315866219