I'm curious to see what others are using for isoelectric focusing gel (IEF) analysis of purified proteins. Previously we had been using the GE PhastSystem with precast semi-dry PhastGel IEF gels, however this system is now discontinued. Many alternative systems that we have been looking into are either too time consuming in comparison, use up too much sample, or have lower quality in data in comparison to the PhastGel.
Briefly, we're interested in comparing pI migration across recombinant and homogeneous protein-susbtrate complexes that vary by -1 or -2 charges.
Thanks.
Without knowing which is best and applicable for you, there are several options to do that.
Capillary electrophoresis (cIEF) or charge variant analysis either by combining UV or MS detection employing pH gradient are very useful and versatile options. Fast, high resolution, practical, and relatively cheaper options...These could be considered like chromatofocusing as the old application of Cytiva (pH gradient on resin, like IPG strips on IEF@2D-PAGE). We also used amphloyte-assisted IPG strips (3-10) PROTEAN® i12™ IEF system to resolve different proteins according to their varying pI without further separation on SDS-PAGE as the 2nd dimension of 2D-PAGE and analyzed via PDquest software. This extends the research indeed. There are also HRMS Native-mass spec applications and integrated deconvolution software that clarify the net charge and causative PTMs along with the charge distribution (envelope pattern)...
Hope this fits with your expectations...
Hi Adam,
The company SERVA is still selling pH 3-9 IEF gels for use with the Phast system (see link below):
https://www.serva.de/enDE/ProductDetails/4247_43366_SERVA_IEF_Gel_3_9_for_PhastSystem_TM_Size_to_50_mm_x_42_mm_x_0_43_mm.html
However, Fisher Scientific is suggesting Invitrogen NOvex IEF Protein Gels, pH 3-10 (Product Code 10741264) as an alternative product.
Best,
Murat
- Badges
- Science topic
- Similar topics
- Papers