Separation of transcriptively active and inactive chromatin. Agarose gel chromatography

Area of Science:

• Molecular Biology
• Chromatin Biology
• Biochemistry

Background:

• Agarose gel exclusion chromatography is a method used to fractionate chromatin.
• This technique is often employed to separate transcriptionally active and inactive chromatin fractions.

Purpose of the Study:

• To investigate the validity of agarose gel exclusion chromatography for separating transcriptionally active and inactive chromatin.
• To identify potential artifacts introduced by the method that could influence experimental outcomes.

Main Methods:

• Fractionation of sheared chromatin using agarose gel exclusion chromatography.
• Analysis of chromatin aggregation under high ionic strength and divalent cation concentrations.
• Assessment of radioactively labeled nascent RNA association with fractionated chromatin.

Main Results:

• Chromatin undergoes non-specific aggregation in the column elution buffer due to high ionic strength and divalent cations.
• This aggregation artifactually locates newly synthesized RNA within the exclusion volume.
• The presence of aggregated chromatin and RNA invalidates assays relying on their co-localization.

Conclusions:

• The agarose gel exclusion method for chromatin fractionation is unreliable.
• Artifactual aggregation compromises the accuracy of assays used to determine chromatin template activity.
• The efficacy of agarose gel exclusion chromatography for separating active and inactive chromatin is highly questionable.