The µMACS FactorFinder Kit facilitates the isolation of virtually any transcription factor from eukaryotic cells, prepared cell lysates, or protein solutions which either directly binds to DNA or forms a complex with other DNA-binding transcription factors. Based on the renowned MACS Technology the µMACS FactorFinder Kit offers a rapid and convenient procedure to isolate high-purity transcription factors. The protocol allows for the isolation of transcription factors in just 1.5 hours. No centrifugation or buffer removal is required.
Detailed procedure
The isolation of transcription factors with a µMACS FactorFinder Kit requires a biotinylated capture DNA that contains the specific transcription factor recognition sequence. After cell lysis the lysate is incubated with the biotinylated capture DNA followed by incubation with µMACS Streptavidin MicroBeads. The sample is loaded onto a µ Column placed in the magnetic field of a µMACS or thermoMACS Separator. After washing, complexes containing magnetically labeled DNA and binding transcription factors are retained on the column. The DNA-protein complex is then eluted from the column using either Elution Buffer or native Elution Buffer.
Downstream applications
Prior to elution of the transcription factor on-column enzymatic reactions, for example the evaluation of potential phosphorylation sites, can be performed using the thermoMACS Separator. Isolated transcription factors can be further analyzed by SDS-polyacrylamide gel electrophoresis, Western blot analysis
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, and mass spectrometry. Analysis of binding specificities for DNA sequence motifs can be performed by native elution conditions where the isolated transcription factors retain their functionality.
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