This helps achieve separation from the faster-moving SDS micelles that interfere with peptide resolution in Tris-glycine buffer systems. Superior resolution is achieved by slowing the migration rate of the peptide-SDS complexes. Tricine gels are ideal for separation of peptides and small proteins with a molecular weight <10 kDa. When loading the protein marker or the protein samples, we suggest using loading tip which can easily get close to the well bottom and therefore minimize the problem of insufficient stacking of very small proteins.ģ. To obtain better stacking of small proteins, 100V/15min followed by 150V/ 60min is worthy of try when running with a 20% TG gel.Ĭ. Adjust voltage and time of electrophoresis. Less than 5 mm (from the well bottom to the top of separation layer) of stacking layer is suggested.ī. The long length of stacking gel will lead to insufficient stacking of very small proteins, and thus causing ambiguous result. If gel with higher than 15% acrylamide is needed to be used, we suggest:Ī. Gel with higher than 15% acrylamide can be used, however, less satisfied resolution of protein separation is frequently observed due to technical difficulty in preparation of gel higher than 15%, including uneven polymerization, bubble formation, and most importantly, the ambiguous results for the small proteins/ peptides. Gel with 15% acrylamide in Tris-Glycine system is usually able to well separate small proteins ranging from 5 to 10 kDa.Ģ. It is concluded that the estimated molecular weight of SMOBIO’s pre-stained marker shows a curve matching well with that of unstained native proteins (MARK12), representing a good estimation of the MW of each pre-stained protein in the SDS-PAGE analysis.ġ. Although it is impossible to define "precision" for molecular weight of proteins in SDS-PAGE, we did compare the migration pattern of pre-stained markers with unstained protein marker (Invitrogen MARK12) for calibration. Therefore, in the product description, we suggest our users to calibrate the MW against their interested proteins. We did compare the migration patterns of SMOBIO’s Protein Markers with other brands, and we concluded that it was difficult to define “precision” due to the reasons mentioned above. For example, a protein which is highly hydrophilic might show a particular higher position in the SDS-PAGE analysis when compared to a hydrophobic one. It is known that the analysis of protein size by an SDS-PAGE is only for “estimation” because of the intrinsic variation of amino acid composition in all proteins including stained and non-stained ones. Biotinylated protein ladder detection pack is designed to detect the molecular weight ladders on Western blots when using the horseradish peroxidase (HRP) based. Usually, pre-stained marker is written on “estimated molecular weight” for caution.
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