Using SAM²® Biotin Capture Membranes to Study Protein:DNA Binding

Introduction

The SAM²® Biotin Capture Membranes can be used to study biotinylated molecules based on their affinity for streptavidin. The SAM^2® Membranes linearly bind substrates and have low nonspecific binding. For DNA:protein binding studies, you have the option of using either biotin-labeled proteins with ³²P-labeled oligos or ³⁵S-labeled proteins with biotinylated oligos. DNA binding analysis is simple with the SAM^2® Membranes. The in vitro translated protein and the appropriately labeled oligo are incubated and then an aliquot is spotted onto the Membrane. The Membrane is washed and each square is counted with a scintillation counter.

Methods

In Vitro Translation Reactions: Fifty-microliter reactions were assembled with either 500ng pSP64polyA-cjun plasmid or no DNA. Reactions were performed in the presence of no label, 2µl [³⁵S]methionine or 1µl Transcend™ tRNA using either the TnT^® SP6 Quick Coupled Transcription/Translation System (#TM045) or the TnT^® Coupled Wheat Germ Extract System (#TB165).

DNA Probe Preparation: The AP1 Consensus Oligo (Cat.# E3201) was 5´-end labeled with [γ-³²P]ATP and T4 Polynucleotide Kinase (Cat.# M4101) following the Gel Shift Assay System Technical Bulletin (#TB110). Biotin 5´-end labeled AP1 Oligos were prepared by annealing a labeled top strand oligo with the complementary bottom strand oligo then verifying the oligo was now double-stranded by polyacrylamide gel analysis (final concentration = 2 pmoles/µl).

SAM^2® Membrane Analysis: Reactions were assembled in a final volume of 15µl and contained 1X Gel Shift Binding Buffer, 1µl labeled AP1 Oligo in the presence or absence of the AP1 protein (3–10µl in vitro translation reactions), cold AP1 oligo (1–3µl; 2pmol/µl), or 1µl cold SP1 oligo. The assembled reactions were incubated on ice for 20–30 minutes, spotted onto SAM^2® Biotin Capture Membranes, washed 3 × 10 minutes in 1X Gel Shift Binding Buffer without poly(dI-dC)/poly(dI-dC), and each square was counted in a scintillation counter.

Results

Figure 1 shows that Transcend™-labeled c-jun proteins expressed in the TnT^® SP6 Quick Coupled Transcription/Translation System or the TnT^® Coupled Wheat Germ Extract System bind to the ³²P-labeled AP1 Consensus Oligo. The presence of unlabeled AP1 Oligo (100-fold excess) significantly reduced the binding of labeled AP1 Oligo to the c-jun protein.

Figure 1. Detection of ³²P-AP1 Oligo binding to Transcend™-labeled c-jun proteins using a SAM²® Membrane.

Duplicate in vitro translation reactions were performed using Transcend™ tRNA and the TnT® SP6 Quick Coupled Transcription/Translation System (RRL) or the TnT® Coupled Wheat Germ Extract System (WGE) in the presence and absence of the pSP46polyA-cjun DNA as described in Methods. An aliquot of each expression reaction (RRL, 6µl and WGE, 10µl) was incubated with ³²P-labeled AP1 Oligos in the presence and absence of unlabeled AP1 Oligos. The reactions were spotted onto SAM²® Membranes and each square was counted in a scintillation counter. The results shown are averages of the duplicates.

To test the specificity of the binding, we performed reactions in the presence of an unlabeled specific competitor (AP1 Oligo) and an unlabeled nonspecific competitor (SP1 Oligo). Both competitors were in 300-fold excess. Figure 2 shows that unlabeled SP1 Oligo is an ineffective binding competitor when compared to unlabeled AP1, demonstrating the specificity of binding detection by the SAM^2® Membranes. Proteins expressed with the TnT^® SP6 Quick Coupled Transcription/Translation or the TnT^® Coupled Wheat Germ Extract Systems had similar specificities.

Figure 2. Specificity of binding of in vitro expressed proteins to ³²P-labeled oligos using the SAM²® Membranes.

In vitro translation reactions were performed using the TnT® SP6 Quick Coupled Transcription/Translation System (RRL) or the TnT® Coupled Wheat Germ Extract System (WGE) in the presence and absence of the pSP46polyA-cjun DNA as described in Methods. Competition reactions were performed using an aliquot of each expression reaction (RRL, 6µl and WGE, 10µl) incubated with ³²P-labeled AP1 Oligos and unlabeled AP1 (specific competitor) or SP1 Oligos (nonspecific competitor). The reactions were spotted onto SAM²® Membranes and each square was counted in a scintillation counter.

Finally, we tested the interaction between a [³⁵S]methionine-labeled in vitro expressed c-jun protein and a biotinylated AP1 Oligo captured on SAM^2® Membranes (Figure 3). Protein expressed with both the TnT^® SP6 Quick Coupled Transcription/Translation System and the TnT^® Coupled Wheat Germ Extract System bound the AP1 Oligo. Due to volume constraints in the binding reaction, only a threefold excess of unlabeled AP1 Oligo was used and competition with the labeled AP1 Oligo was incomplete.

Figure 3. Binding of in vitro expressed ^[35S]methionine-labeled proteins to a biotinylated AP1 Oligo and SAM²® Membrane.

Duplicate in vitro translation reactions were performed using [³⁵S]methionine and the TnT® SP6 Quick Coupled Transcription/Translation (RRL) or TNT® Coupled Wheat Germ Extract (WGE) Systems in the presence and absence of the pSP46polyA-cjun DNA as described in Methods. An aliquot of each expression reaction (RRL, 6µl and WGE, 10µl) was incubated with biotinylated AP1 Oligos in the presence and absence of unlabeled AP1 Oligos (6pmol). The reactions were spotted onto SAM²® Membranes and each square was counted in a scintillation counter. The results shown are averages of the duplicates.

Conclusion

The detection of DNA binding does not require gel analysis. DNA:protein interactions can be measured using either a Transcend™-labeled protein and a ³²P-labeled oligo or a ³⁵S-labeled protein and a biotinylated oligo in conjunction with the SAM^2® Membranes. SAM^2® Membrane analysis is easier than gel analysis, especially if large numbers of samples will be analyzed.