I have been trying to purify a transcription factor. Protein has MBP and His Tag. I use His TRAP as my first purification step and then after I do size exclusion, but my protein (83 KDa) elutes just after the void volume in SD 200pg. and when I analyze through the SDS it elutes with a bunch of proteins (SDS page given below). How can I prevent this?
I also tried Mono Q after size exclusion, but the impurities remain. I cannot use the amylose column as my protein precipitates once I try to elute protein with 10 mM Maltose (I tried batch-wise purification with buffer 50 mM Tris HCl ph 7.5, 500 mM NaCl, 15% glycerol, 10 mM Maltose still it precipitates, so I avoided using the amylose column)
The buffers I use are lysis buffer – 50 mM Tris HCl ph 7.5, 500 mM NaCl, 10% glycerol, Lyzozyme, DNAse, and PMSF.
HisTRAP buffer- 50 mM Tris HCl ph 7.5, 500 mM NaCl, 10% glycerol eluted with a gradient of 500 mM Imidazole
Size exclusion buffer - 50 mM Tris HCl ph 7.5, 250 mM NaCl, 10% glycerol
Use a column with the right pore size and a mobile phase that is compatible with the protein. Use a gradient elution to seperate impurities from proteins and desalting step to remove salt. Finally, a column guard with size exclusions.
Size exclusion chromatography is a widely used technique for protein purification, but one of its main challenges is preventing the co-elution of proteins with other impurities. To avoid this problem, it is essential to optimize sample preparation, choose the appropriate column, and run the column at the appropriate flow rate. One of the most important factors is to avoid overloading the column. Overloading can lead to peak broadening, reduced resolution, and protein co-elution. By using a larger volume column or lower sample concentration, overloading can be avoided. It is also beneficial to use buffer additives such as salt, detergent, or organic solvents to optimize the separation of proteins. If the problem persists, other chromatographic techniques such as ion exchange chromatography, hydrophobic interaction chromatography, or affinity chromatography can be used to purify the protein. Analyzing fractions with SDS-PAGE or other analytical methods can help to determine which fraction contains the protein of interest. Overall, preventing protein co-elution requires careful optimization of the purification protocol, column selection, and chromatographic conditions to obtain pure protein samples.
I assume you have a MBP fusion protein because you had solubility problems without? I have seen several examples of proteins behaving like you describe: The MBP fusion protein is soluble but migrates as very large aggregates in SEC. A protein of 83 kDa should migrate well inside the fractionation range of Superdex 200. Most likely you protein of interest is still not correctly folded and aggregates, but the aggregates are kept in solution by correctly folded MBP. Have you considered denaturing and refolding? You may also try adding urea at concentrations that do not denature the protein - that can help "loosening up" the aggregates and perhaps release some of the impurities that seem trapped in there. Keep the urea concentration below 2 M.
There are several methods available to enrich for low abundance TFs, including TFs present at less than 105 copies per cell. Because all purifications rely on a specific assay for the protein of interest, the assays used must be first discussed. The most popular method to determine whether a TF binds to particular DNA sequence is the electrophoretic mobility assay (EMSA), also called a gel shift assay. This procedure uses either radio- or fluorescence-labeled DNA, incubated with pure or crude (nuclear) extract prior to resolution with native (non-denaturing), polyacrylamide gel electrophoresis (PAGE), to detect TF-DNA complexes. If a TF is present and binds the labeled DNA, then the corresponding TF-DNA complex will be detected from its slower migration on the gel (I'd read article about that doi: 10.1002/mas.21369)
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