Discover your next important protein interaction with this new Pierce Far-Western Kit

ProFound Far-Western Biotinylated-Protein:Protein Interaction Kit

The first nonisotopic far-Western Protein:Protein Interaction Kits that detect interactions in-gel or on-membrane.

Far-Western protein:protein interaction analysis need not be difficult or mysterious. The new ProFound Far-Western Biotinylated-Protein:Protein Interaction Kit represents a novel nonisotopic approach to far-Western analysis that can be performed either directly in the gel or, like the classical method, on a membrane using sensitive chemiluminescent detection.

Far-Western Primer

A far-Western is a method designed to detect protein interactions. In a classical far-Western strategy, proteins are first transferred from gel to membrane. These proteins are then probed with a pure protein known to interact or putatively interact with one or more proteins transferred to the membrane. The protein probe or "bait" protein is isotopically labeled and the interaction with the "prey" protein is detected on the membrane. Since the probe in far-Western analysis is not an antibody, as it would be in classical Western blot detection, the term "far" was adopted to make this distinction. Therefore, the term far-Western emerged to describe a very useful in vitro protein:protein interaction method.

ProFound Far-Western Protein:Protein Interaction Kit

A Nonisotopic Approach to Far-Western Analysis In-Gel or On-Membrane

How does this far-Western kit work?

Exclusive Pierce UnBlot Technology

Adopted for Detection of Protein Interactions

The far-Western protein:protein interaction kit incorporates the major advantages inherent in the Pierce UnBlot In-Gel Chemiluminescent Detection Platform, including the subsequent elimination of the need for protein transfer. Interactions can be directed directly in-gel with the appropriate probe. In addition, methodology is provided that enables use of the kit for on-membrane applications if additional sensitivity is needed to detect the interaction of interest. Optimization may be required to obtain the best results in a given system.

Kit Applications

· Protein:Protein Interaction Discovery

Direct in-gel or on-membrane detection of a potential interaction using a purified biotinylated-protein as a probe

Compared to classical far-Western detection on a membrane, the in-gel technology incorporated into this kit is a faster and easier method to reveal potential interactions.

In those instances where an interaction cannot be readily detected in-gel, the reagents provided with this kit can also be used for on-membrane (nitrocellulose/PVDF) detection.

The strategy employed aids in the detection of strong to moderate interacting pairs. Weak or transient interactions may not be readily observed with these probes either in-gel or on-membrane.

· Protein:Protein Interaction Confirmation

The far-Western based kit can be used as an in vitro tool for the verification or confirmation of known or putative bait:prey interacting pairs.

Far-Western Biotinylated-Protein:Protein Interaction Kit Results

Confirmation of a Known Protein Interactions with a Biotinylated Bait.

In-gel detection using UnBlot Methodology vs. classical on-membrane detection

Figure 1. Protein:Protein Interaction – Far Western In-Gel and Membrane. Purified BIR2 and BIR2 bacterial lysate samples were separated by SDS-PAGE on 4-20% Tris-Glycine gels. Two gels (1A and 1B) were probed and processed using in-gel far-Western detection and UnBlot Technology-based method. A third gel was transferred to a nitrocellulose membrane and processed using the classical far-Western membrane-based probing and detection. Gel 1A) The samples were probed in-gel with purified Biotinylated-Smac diluted 1:100 followed by Streptavidin-HRP diluted 1:20,000.

Gel 1B) The control gel was probed with non-biotinylated purified Smac followed by Streptavidin-HRP. Gel 1C) Following the in-gel far-Western detection, one gel was stained for total protein with GelCode Blue Stain Reagent (Product # 24590). Gel 1D) The membrane was probed with purified Biotinylated-Smac diluted 1:200 followed by Streptavidin-HRP diluted 1:250,000.

Pierce UnBlot Chemiluminescent Substrate Working Reagent was used for the detection of 1A, 1B and 1D shown above. Lane 1 and 2 correspond to purified BIR2, 1.7 µg and 3.1 µg, respectively. Lane 3 corresponds to BIR2 bacterial lysate. The biotinylation of Smac was done using Sulfo-NHS-LC-Biotin (Product # 21335). BIR2 (GST-BIR2)- Binding Domain of XIAP Smac (9xHis-Smac)- Second Mitochondrial-Derived Activator of Caspases

Figure 2. Chaperone Proteins: Interaction of p90 with Biotinylated-Hop. Chaperone protein p90 (denatured) was separated by SDS-PAGE on a 10-20% Tris-Glycine gel. Lane 1, 2 and 3 correspond to 15 µg, 30 ng and 15 ng, respectively, of p90 chaperone protein. Gel 2A) In-gel far-Western detection. Gel 2A was probed with Biotinylated-Hop followed by Streptavidin-HRP. Gel 2B) In-gel far-Western detection control. Gel 2B was probed with non-biotinylated Hop followed by Streptavidin-HRP. Gel 2C) Total protein staining. Following the in-gel immunodetection procedure in 2A and 2B above, one gel was stained with GelCode Blue Stain Reagent, a Coomassie Dye-based reagent. Gel 2D) Membrane far-Western detection. A third gel was run and transferred to a nitrocellulose membrane. The membrane was probed with Biotinylated-Hop followed by Streptavidin-HRP. The protein was detected with UnBlot Substrate Working Reagent. The biotinylation of Hop was done using Sulfo-NHS-LC-Biotin (Product # 21335).

Figure 3. Probing for interactions in-gel with a biotinylated whole cell lysate. Mycobacterium avium complex (MAC101) lysate samples were separated by SDS-PAGE on two 8-16% Tris-Glycine gels (3A and 3B). Lanes 1, 2 and 3 correspond to 5 µg, 30 µg and 15 µg of total MAC101 lysate protein, respectively. Gel 3A) Gel was incubated with a 1:100 dilution of Biotinylated-Human epithelial cell lysate (B-Hep2 cell lysate). Starting protein concentration was estimated at 2 mg/ml. Gel 3B) Negative control gel was incubated in the appropriate dilution buffer alone. Gels 3A and 3B were developed using Streptavidin-HRP (at a 1:25,000 dilution of a 1 mg/ml solution) and UnBlot Substrate Working Reagent.

1. Towbin, H., Staehlin, T. and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354.
2. Renart, J., Reiser, J. and Stark, G.R. (1979). Transfer of proteins from gels to diazobenzyloxymethyl paper and detection with antisera. A method for studying antibody specificity and antigen structure. Proc. Natl. Acad. Sci. USA 76, 3116-3120.
3. Burgess, R., Arthur, T.M. and Pietz, B.C. (2000). Mapping protein-protein interaction domains using ordered fragment ladder far-Western analysis of hexahistidine-tagged fusion proteins. Meth. Enzymol. 328, 141-157
4. Lin, W. and Kasamatsu, H. (1983). On electrotransfer of polypeptides from gels to nitrocellulose membranes. Anal. Biochem. 128, 302-311.
5. DenHollander, N. and Befus, D. (1989). Loss of antigens from immunoblotting membranes. J. Immunol. Meth. 122, 129-135.
6. Reddy, V.M. and Kumar, B. (2000). Interactions of Mycobacterium avium complex with human respiratory epithelial cells. J.I.D 181,1189-1193.

Ordering Information

Instruction Book   MSDS

Buy Product # Description Pkg. Size Files Price Add 23500 ProFound Far-Western Biotinylated-Protein:Protein Interaction Kit Streptavidin-Horseradish Peroxidase (Streptavidin-HRP), 0.1 mg, lyophilized Dilution Buffer (10X), 50 ml BupH Phosphate Buffered Saline Packs, 17 packs, 0.1 M phosphate, 0.15 M NaCl, pH 7.2 UnBlot Luminol Enhancer, 55 ml UnBlot Stable Peroxide, 55 ml Surfact-Amps 20, 6 x 10 ml vials, 10% solution Pre-Cut Cellophane, 10 sheets kit $335.00