Autoantibody research using the Surf-Blot System

August 29, 2013

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Duke University Medical Center
Associate Professor
Company: Idea Scientific
Product Name: Surf-Blot System
Catalog Number: 5006

 

 

 

It is well known that malignant cells can trigger both cellular and humoral immune responses. Our laboratory focuses on the humoral response in lung cancer through investigation of autoantibodies, which are antibodies against naturally occurring proteins or nucleic acids in the body. It is our belief that the specific targets to which the humoral immune response is directed can provide information that may be of value in the search for new diagnostic and therapeutic strategies. In our early studies, we sought to catalog the humoral immune response in dozens of lung cancer patients. To accomplish this, we relied heavily on immunoblots. The problem we encountered with this method, however, was that conventional immunoblots work best when one antibody or one serum sample is to be tested and not when the goal is to investigate the immune response in many patients. One approach that is frequently used to circumvent this limitation is to cut the protein-containing blot into thin strips and probe each strip with a different serum specimen. Although functional, this method is labor intensive and impractical. The Surf-Blot System from Idea Scientific permits a single blot to be probed with as many as 33 different serum specimens without having to cut the blot into strips. Without this device, much of our research would have been dead in the water.

Experimental Design and Results Summary

Application

Immunoblot

Starting Material

Detergent-solubilized protein extracts from immortalized cells or tumor tissue.

Protocol Overview

Proteins are separated via SDS-PAGE and blotted to PVDF. For use with the Surf-Blot System, gels designed for the second dimension of 2D PAGE should be used. These are often referred to as IPG + 1 gels since they have one large well for the IPG strip and one small well for the MW ladder. The PVDF is then blocked for one h in PBS containing 0.1% (v/v) Tween-20 and 5% (w/v) non-fat dry milk (PBST-milk). The blocked membrane is then transferred, dripping wet, into the Surf-Blot apparatus as described. The apparatus is assembled and clamped shut with bulldog clips with the assembled apparatus propped up at about a 45° angle. Diluted serum is pipetted into each channel opening. The apparatus is then placed horizontally on a rocking platform and rocked for 2 h at room temperature. Holding the apparatus over the sink, the diluted serum specimens are removed by squirting PBST through each channel using a squirt bottle. The apparatus is disassembled and the blot is washed 4 x 5 min in PBST. At this point, the rest of the protocol (i.e. secondary antibody, wash, chemiluminescent substrate incubation, X-ray film exposure) is identical to that for a conventional immunoblot.

Tips

1) The metal plate that serves as the base of the Surf-Blot apparatus is curved. The apparatus must be assembled with the convex face of the curvature facing up in order to avoid leakage between the channels. 2) When clamping the apparatus together with bulldog clips, start at one end and systematically (and symmetrically) move toward the opposite end. This will ensure that the apparatus is evenly clamped and no leakage will occur. 3) After incubation with primary antibodies, flush out the channels with wash buffer before disassembling the apparatus to avoid cross-contamination among the channels. 4) Thoroughly clean all parts of the Surf-Blot apparatus as soon as you can. I do it during the washes after the primary antibody. Let everything air dry before storing. 5) For storage, place a Kim Wipe between the rubber pads to prevent them from sticking together.

Results Summary

The results, which in our case is a developed piece of X-ray film, look similar to a conventional immunoblot made from a gel containing multiple lanes of separated proteins from different sources (see image). Different patterns of immunoreactive bands are observed in each lane but, in the case of the Surf-Blot, each lane was probed with a different primary antibody (or serum specimen).

Features Summary

Permits anywhere from 21 to 33 different primary antibodies or sera to be simulateously tested via immunoblot.

Additional Notes

Idea Scientific has several different sizes of Surf-Blot Systems to accomodate blots from essentially any size gel. Also, the design of the apparatus permits fewer than the total number of channels to be used.

Image Gallery

Summary

The Good

The Surf-Blot System is without a doubt the simplest and most effective means to perform immunoblots with multiple primary antibodies or serum specimens simultaneously.

The Bad

Although the Surf-Blot System permits experiments to be carried out that would otherwise be very difficult if not impossible, assembly, disassembly, and cleaning the apparatus is a bit cumbersome. This is especially evident when optimizing for different primary antibodies when several runs may be required. Also, only one secondary antibody can be used on the resulting blot, unless the blot is cut after incubation with primary antibody.

The Bottom Line

This is a very useful and economical piece of equipment. Given the care needed to use the apparatus successfully, it may be best to appoint someone to be the Surf-Blot guru rather than have everyone in the lab using it.

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