Overview

Western blot analysis is essential in studying specific proteins in a mixture of protein samples. For example, it can determine the presence of a protein in different tissues at different development stages or the protein’s association with other proteins under different physiological conditions.^1,2,3 For Western blot analysis, polyacrylamide gel electrophoresis (PAGE) is used to separate proteins in the sample based on the molecular weight of individual protein subunits under denaturing conditions or the combined size and shape of protein complexes under native conditions. For the former, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is suitable for analyzing crude extracts because of its tolerance for variations in the salt content and pH of the protein sample solution. After SDS-PAGE, proteins are transferred onto a membrane, and the target protein(s) on the membrane are detected with specific antibodies.⁴

Blood plasma occupies over 50% of total blood volume and contains ions, nutrients, waste, and various proteins. Among those proteins, albumin is mammals’ most abundant protein. It is made in the liver and released into the bloodstream.⁵ It modulates plasma oncotic pressure for properly distributing body fluids between blood vessels and tissues. With a high binding capacity, albumin is also the essential transporter for various metabolites, drugs, nutrients, metals, and other molecules. Likewise, bovine serum albumin (BSA) is critical in maintaining oncotic pressure within capillaries and transporting essential molecules in cows.⁶ Due to its abundance and affordability, BSA has been used in a wide variety of lab applications, including the blocking reagent in a Western blot and a common protein for constructing standard curves in protein quantification.

Unlike mammals with a closed circulatory system, fruit flies (Drosophila melanogaster) have an open circulatory system with body fluid (hemolymph) in the body cavity and appendages.⁷ They have a simple tube-like “heart” pumping the hemolymph from the posterior towards the anterior by a dorsal vessel to connect the heart and the aorta.⁸ Oxygen is distributed by a network of tracheal tubes open to the outside through spiracles in the fly.⁷ The hemolymph, so-called “blood,” represents the interstitial fluid, and its role is to transport nutrients throughout the body and waste products for excretion. Although insects do not have erythrocytes, hemoglobins are expressed in 29 orders of insects.⁹ The fruit fly has a hemoglobin monomer of ~17 kD.¹⁰ Its synthesis is associated with the tracheal system and body fat. However, this 17 kD hemoglobin is not exported into the hemolymph. Therefore, in addition to simple diffusion, the Drosophila oxygen supply relies on hemoglobin-mediated oxygen transport and storage but not hemolymph. The function of hemoglobin-like proteins is analogous to mammalian oxygen transport.

Insect hemolymph contains circulating hemocytes and transports hormones, nutrients, and metabolites. Is serum albumin or its ancestor protein(s) present in the hemolymph of fruit flies with an analogous function as the transporter of essential molecules in mammals? For this lab module, hemolymph proteins are isolated from fruit fly larvae. Bovine calf serum (BCS), fetal bovine serum (FBS), and the isolated protein sample are quantified by Bradford assay and prepared in Week 1, followed by SDS-PAGE and Western blotting in Week 2, and concluded with immunodetection with anti-BSA antibody in Week 3. The procedure is summarized below (Figure 5-1).