The DNAse foot-printing method starts by focusing on a given region of interest (e.g. a promoter region) and amplifying it by PCR to obtain lots of sample. It then throws in the TF and then the DNAse. The mix is left to stir for a short time and then gel electrophoresis is run to compare the pattern of fragments in a control (no TF) and in the sample. If the TF has bound the sample, it will have protected a stretch of DNA (encompassing some fragments of the control) and thus those fragments will not appear in the sample gel. The fragments can then be cut-out from the gel, purified and sequenced to obtain the sequence of the protected region. This is often used to identify the binding motif of a TF for the first time. The foot-printing will typically resolve the protected region down to 50-100 bp, and the sequence can be then examined for possible TF-binding sites either by eye of using a computer search.

In-vitro transcription is often used to validate that a particular TF can or cannot activate/repress a gene in the absence of other proteins that are present in in vivo conditions.

This is a technique used to detect typically mRNA with greater precision than Northern blot or RT-PCR. Therefore, it is commonly used to assess gene expression (specifically transcription). RNAse protection uses labelled RNA probes to bind desired targets. RNAses are then added to the mix and degrade all RNA that is not bound to probes. The remaining RNA is typically run on a gel to detect the size (and label) of the probe and determine which RNA it is.

Visual inspection of promoter region to identify putative binding sites based on previous knowledge

The DNAse foot-printing method starts by focusing on a given region of interest (e.g. a promoter region) and amplifying it by PCR to obtain lots of sample. It then throws in the TF and then the DNAse. The mix is left to stir for a short time and then gel electrophoresis is run to compare the pattern of fragments in a control (no TF) and in the sample. If the TF has bound the sample, it will have protected a stretch of DNA (encompassing some fragments of the control) and thus those fragments will not appear in the sample gel. The fragments can then be cut-out from the gel, purified and sequenced to obtain the sequence of the protected region. This is often used to identify the binding motif of a TF for the first time. The foot-printing will typically resolve the protected region down to 50-100 bp, and the sequence can be then examined for possible TF-binding sites either by eye of using a computer search.

In-vitro transcription is often used to validate that a particular TF can or cannot activate/repress a gene in the absence of other proteins that are present in in vivo conditions.

This is a technique used to detect typically mRNA with greater precision than Northern blot or RT-PCR. Therefore, it is commonly used to assess gene expression (specifically transcription). RNAse protection uses labelled RNA probes to bind desired targets. RNAses are then added to the mix and degrade all RNA that is not bound to probes. The remaining RNA is typically run on a gel to detect the size (and label) of the probe and determine which RNA it is.

Visual inspection of promoter region to identify putative binding sites based on previous knowledge