UnBlot In-Gel Chemiluminescent Detection Kit—Rabbit From Pierce Biotechnology

The UnBlot In-Gel Chemiluminescent Detection Kit is a substitute for the Western blot technique. In Western blotting, the transfer of proteins is required after the separation of protein bands on a gel; the success of the transfer is dependent on the size and nature of each protein. Because the UnBlot In-Gel Chemiluminescent Detection Kit eliminates the transfer step, it is especially beneficial for proteins which do not transfer efficiently. Using this kit, all proteins are detected directly in the gel. The gel can be stained for the quantification of the total protein even after incubating with primary and secondary antibodies.

Following a pre-treatment step, this kit utilizes a highly sensitive chemiluminescent substrate for horseradish peroxidase (HRP). The UnBlot In-Gel Chemiluminescent Detection Kit contains UnBlot Substrate, Stabilized Goat Anti-Rabbit-HRP conjugated antibody (10µg/ml), dilution buffer (10x), BupH Pack (17 PBS Buffer packs, 0.1 M phosphate, 0.15 M NaCl, pH 7.2), Surface Surfact-Amps™-20 (5 x 10 ml vials), Hands-Off™ Incubation: Colander 1 along with two trays, CL-XPosure Film (5 x 7), UnBlot Luminol Enhancer, UnBlot Stable Peroxide, and pre-cut cellophane. This kit contains all the reagents to perform at least 10 mini blot gel detections.

If the polyacrylamide gel is poured using the AquaSil™ Siliconizing Fluid (Product No. 42799, Pierce Biotechnology) as recommended, there is no need to add sodium dodesyl sulfate (SDS) to the gel for polymerization. After electrophoresis, the polyacrylamide gel is taken out of the gel cassette and the stacking gel is carefully removed with a knife, without touching the gel. The gel is then transferred to the Hands-Off™ Incubation Colander. The gel can be treated with 50% isopropyl alcohol or with 50% methanol if AquaSil™ Siliconizing Fluid was used; following treatment, the gel is washed with ultrapure water. Then gel is then incubated in the primary antibody specific for our protein of interest (final antibody concentration is 0.1-2.0 ug/ml) for 1 hour followed by washing in PBS-T solution. The secondary antibody (provided with the kit) incubation follows for 1 hour. The secondary antibody is generally used at a dilution of 1:500-1:1000; the exact dilution will depend upon the nature and quantity of the protein being detected. After washing with PBS-T, the gel is incubated in the UnBlot Substrate Working Solution for 5 minutes. The UnBlot Stable Peroxide is mixed with the UnBlot Luminol Enhancer to prepare the UnBlot Substrate Working Solution freshly just before the use and any exposure to light should be avoided. Finally, the gel is rinsed with ultrapure water and is put between sheets of cellophane and exposed to a CCD camera or film.

Proteins from 20,000-160,000 kd can be screened efficiently using this kit. There are lots of advantages to using this product: Since there is no transfer step, there is no artificial change in the amount of antigen present, reprobing can be done efficiently, blocking is not required, it can detect protein down to 1ng of protein and thus, is compatible with ECL substrate, and total protein can be visualized even after the immunostaining. Another advantage to this kit is that antigens remain in their native form. This is contrast to classical Western blotting which requires protein denaturation. Denaturation of the epitopes against which the primary antibody was raised can sometimes cause difficulties in detection. The only disadvantage of this kit is that extra care is needed to deal with the gel as touching the gel may give some background. Otherwise, we can probe the same gel using different antibodies as we do in Western blotting by using Pierce’s stripping buffer (Product # 21059). Furthermore, the same gel can be washed and stained with Gel Code Blue Protein Stain Reagent (also from Pierce) to detect the total protein present in gel.

The aim of my study is to explore the mechanism of cancer chemoprevention by dietary constituents. Multiple drug resistance (MDR) is the major hurdle of cancer chemotherapy. We initially used Western blot analysis to study changes in MDR protein expression, but improper transfer made MDR expressing screening difficult. However, after using Pierce’s Un-blot Kit, I was able to clearly analyze expression levels on the gel itself.

I, therefore, strongly recommend the use of this kit whenever the transfer step is difficult and incomplete.