A novel method that improves sensitivity of protein detection in PAGE and Western blot

Abstract

We have developed a simple and inexpensive method that improves sensitivity of protein and antigen detection in standard PAGE procedures. Our technique uses a sample microloader device with a funnel-like structure, filled with a 4% stacking gel. When attach to the top of a polyacrylamide slab gel, the proteins in a sample are concentrated by electrophoresis into a small volume as they emerge from the device's narrow outlet. Our microloader has several advantages over previous devices, including simple assembly, high versatility, and absence of cross-contamination between lanes. Addition of this device to a slab gel results in a fivefold increase in the sensitivity of antigen detection in a Western blot. As a result, less protein is required for loading and signal detection. Our protocol is a straightforward modification of a standard experimental technique, and is especially useful when only limited sample quantities are available.

Figure 1. Microloader device preparation

Fine tip transfer pipettes (#233, #235, Samco Scientific) were used for the assembly of sample microloaders. Pipette tips were cut to obtain an outlet passage length of 1 cm and a reservoir length less than 1 cm and a 4% polyacrylamide stacking gel was introduced through the outlet passage by capillary action. After stacking gel polymerization, sample microloaders were firmly inserted into a 1 mm-thick acrylamide gel and the reservoir was loaded with running buffer. Special care was taken to avoid bubbles and to ensure close contact between microloaders and gel. A total of 1-15 μl of sample was loaded into each microloader, and electrophoresis was performed at a maximum of 100 V until samples entered into the gel, after which voltage could be increased up to 200 V.

Figure 2. Sensitivity and linear range of western blot with microloaders

A) Representative western blot showing FAK protein detection in a single 15-well 4-15% minigel with and without use of a microloader. This figure illustrates comparison within the same gel between samples loaded directly onto wells versus samples loaded onto microloaders prefilled with a 4% stacking gel that were attached to adjacent wells. Sequential 2-fold dilutions of protein extracts from a E12 embryonic mouse were loaded and FAK signal was visualized using SuperSignal West Dura Chemiluminescent Substrate. B) The films were scanned, and ImageJ software was used to obtain densitometry plots and to quantify signal intensity. C) Densitometry results are plotted into a graph to better show sensitivity and linear range comparison. D) Comparison of FAK signal detection sensitivity in 15-wells and microloader systems in the same gels. Data are obtained from 3 independent experiments and expressed as mean ± SEM.