I am trying to isolate (unknown) proteins from a homogenized cell sample using phage (tomlinson library I+J) however the phage amounts we can create are too low to detect then isolate any protein which may be bound to our phage. So far I have tried his-trap columns and biotinylation to purify out the phage/protein complexes but phage numbers are so low that bound protein does not show up on SDS-PAGE.
Any suggestions?
thanks,
James
Try Silver stain for your SDS page it is quite sensitive, if a protein is there you should see it.
Hi James,
Having some clue of the protein function could help a lot. For instance, if you know it has a protein domain homolog to some enzyme family/ functional protein can indicate a similar function. Based on this, you could develop/look for biochemical detection assays.
If you only have the protein sequence, you might want to perform some LC/MS to isolate and identify the protein; however, it is very likely to denature the protein after the process. Anyway, it is a good start for unknow protein identification based only on its primary sequence.
Cheers,
Fabio
Hello James,
What I would recommend is using orthogonal multi-dimensional liquid chromatography for the targeted isolation of target proteins. Our paper in Garbis, et al., Analytical Chemistry 2011 is based on this principle. Typically, we start with High-Performance Size Exclusion Chromatography for the initial segragation of a protein with a known MW using about 1 mg - 100 ug of the starting protein extract. We then dialysis purify this segment and lyophilize to dryness at 4 degrees Celcius. We then reconstitute in MP and subject to C18 reverse phase chromatography using a 200m X 4.6 mm X 500-1000 A posre size, 5 um particle dimension. We fractionate the target protein to which we know the RT (from using a recombinant target protein standard). If need be, we run this 3-4 more times to enrich its concentration using the material collected form HP-SEC. The purity will be around 50-60% miminimum. We then lyophilize to dryness the collected fraction and subject it to Hydrophilic interaction chromatography (HILIC) using an analogous geometry with RP. The resultng purity will be a minimum of 85%, which is condusive to both bottom-up LC-MS Proteomics for proteins > 50 kDa, or top down LC-MS proteomics for proteins < 50 kDa.
Alternatively, HPLC with gel based approaches can be used for targeted proteins followed by top-down LC-MS proteomics. Please consult with our Bouchal et al, 2013 paper in Proteomics.
- Badges
- Science method
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Methods
- Protein Purification Techniques