using unstained protein ladder for western blot

Doucet and Beauregard produced a protein ladder by disulfide crosslinking a 11 kD designer protein via oxidation in solution 1. There are a few reported examples of home-made protein ladders. These improvements have come at an increased expense with most commercially available unstained ladders costing about US$ 1.00 per lane. 25, 50 kD) and with optional features such as prestaining with dyes for visibility during electrophoresis and on Western blots. Such ladders have been replaced more recently by recombinant ladders with rounded molecular weights (e.g. These native protein ladders were commercially available and relatively inexpensive at about US$ 0.10 per lane. As such, protein ladders constitute critical reference reagents when expressing, purifying or analyzing proteins.Įarly protein ladders were comprised of readily available proteins such as lysozyme (14 kD), soybean trypsin inhibitor (21 kD), carbonic anhydrase (31 kD), ovalbumin (45 kD), serum albumin (67 kD) and phosphorylase b (97 kD). They provide molecular weight standards to estimate the size of proteins separated by gel electrophoresis like SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Protein ladders or molecular weight markers are among the most commonly used reagents in biochemistry experiments. coli and affinity protein purification, and to research laboratories desiring positive controls for recombinant protein expression and purification. These Penn State Protein Ladder expression plasmids also constitute useful reagents for teaching laboratories to demonstrate recombinant expression in E. 50 ml of culture is sufficient to produce 20,000 lanes of individual ladder protein or 3750 lanes of each set of coexpressed ladder proteins. For more efficient production, we have created two polycistronic expression vectors which coexpress the 10, 30, 50, 100 kD proteins or the 20, 40, 60, 80 kD proteins. We have also constructed plasmids to express 150 and 250 kD proteins. Each protein migrates appropriately on SDS-PAGE gels, is expressed at very high levels (10–50 mg per liter of culture), is easy to purify via histidine tags and can be detected directly on Western blots via engineered immunoglobulin binding domains. The system includes plasmids which express 10, 15, 20, 30, 40, 50, 60, 80 and 100 kD proteins in E. Proteins (0.02 to 0.05mg/mL of each) in 62.5mM Tris-H 3PO 4 (pH 7.5 at 25☌), 1mM EDTA, 2% SDS, 100mM DTT, 1mM NaN 3, 0.01% bromophenol blue and 33% glycerol.We have created the Penn State Protein Ladder system to produce protein molecular weight markers easily and inexpensively (less than a penny a lane). Quality tested – each lot evaluated by SDS-PAGE and Western blotting Tagged – each protein in the ladder contains an integral Strep-tag™ II Sequence Reference band – 50kDa band is has greater intensity for easy orientation

Sharp bands – excellent accuracy for detection in with coomassie or silver stains Ready-to-use – supplied in a loading buffer for direct loading on gels Size range – 14 proteins spanning 10 to 200kDa The 50kDa protein band is of greater intensity and serves as a reference band. The Ladder produces sharp bands on SDS-polyacrylamide gel following staining of the gel with Thermo Scientific Pierce Gel Stains or other coomassie dye or silver stains. Thermo Scientific™ Unstained Protein Molecular Weight Marker is a mixture of seven native proteins (14.4 to 116kDa) for use as size standards in protein electrophoresis (SDS-PAGE).Įach protein in the ladder contains an integral Strep-tag™ II Sequence which can be detected directly on Western blots using a Strep-Tactin™ Conjugates or an antibody against Strep-tag™ II Sequence.

Using unstained protein ladder for western blot