Generally, enough 50 µL aliquots are prepared for 2 weeks of experiments and the rest discarded in liquid waste.ĨThe pcv is approximately 4 mL (from ten of 182 cm 2 cell culture flasks) and contains 4 × 10 8 cells.ĩLong dialysis is detrimental to activity. To this would be added 3 mL 10 nM unlabeled oligonucleotide (in TE) to give a total of 5 mL and 4 × 10 6 cpm/mL. In this case, the 50 µL labeled oligonucleotide would be diluted to 2 mL with TE, resulting in 10 nM oligonucleotide. The Eppendorf tube used for collection can be pre-weighed and weighed again after collection to determine the volume more accurately.ħIf labeling efficiency (Subheading 3.2, step 5) is 50 %, after desalting the DNA will be 50 µL of 400 nM oligonucleotide, and contain 20 × 10 6 cpm. Each of these SWB techniques provides alternatives to investigate regulatory transcription factors in vitro.Ĭentrifuge the column and collect the eluate containing labeled DNA into a clean 1.5 mL centrifuge tube.ĦDesalting removes over 90 % of the remaining γ- 32P-ATP. Additionally, a method has been developed that allows multiple reprobes with different oligonucleotides on one blot. Coupled with proteomic techniques, several transcription factors have been identified. 1D-SWB uses separation on a single SDS-PAGE dimension, 2D-SWB uses separation by isoelectric focusing followed by SDS-PAGE in the second dimension, while 3D-SWB uses separation by DNA gel shift on a non-denaturing gel before 2D-SWB. In this chapter, three detailed SWB methods are described that differ in the number of electrophoresis dimensions used for protein separation. Since the original SWB assay, first described in 1980, many extensions of the SWB method have been developed. Oligonucleotides are generally 20 bp in size containing one response element or using core promoter DNA Protein samples are either prepared from nuclear extract or purified by other techniques. 1.įlow chart of SWB experimental procedure. An experimental procedure is shown schematically in Fig. This technique is especially useful in the identification of transcription factor as it gives information on the molecular weights of all DNA-binding proteins involved with a particular sequence of DNA. Any unbound DNA is washed away, and then bands on the blot are detected by autoradiography. To detect the DNA-binding proteins, the proteins are partially renatured and bound to nanomolar concentrations of radiolabeled DNA. The gel containing the separated proteins is electro-transferred (blotted) to a membrane, such as nitrocellulose (NC) or polyvinylidine difluoride (PVDF).

SWB, similar to other blotting techniques, separates proteins (or DNA) by gel electrophoresis. Southwestern blotting assay (SWB) is one of the most powerful techniques to explore protein-DNA interaction and transcription factor regulation. Identification of transcription factors specific to a particular gene is not only significant to gene regulation but also to understanding gene function. Transcription factors are DNA-binding proteins that interact with unique response elements at the promoter region of DNA, or other functional cisacting response elements, resulting in either gene expression or repression. The gene regulatory mechanism involves distinct 6–8 bp DNA motifs referred to as response elements, which bind transcription factors. Regulation of gene expression is essential in human development as well as pathogenesis.

South eastern blotting