![]() ![]() ![]() The traditional method to visualize protein ladders on Western blots is to use prestained protein ladders which remain visible when transferred to the blotted membrane. Other home-made protein ladders have tackled the problem of detecting protein ladders on Western blots. 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. Do not boil.We have created the Penn State Protein Ladder system to produce protein molecular weight markers easily and inexpensively (less than a penny a lane). Thaw the ladder either at room temperature or at 37-40☌ for a few minutes to dissolve precipitated solids. The quality of the BLUeye Prestained Protein Ladder is tested on a lot-to-lot basis to ensure consistent product quality.īLUeye Prestained Protein Ladder Protocolġ. Sizing of proteins on SDS-PAGE and Western blots.Īpproximately 0.1~0.4 mg/ml of each protein in the buffer (20 mM Trisphosphate, pH 7.5 at 25☌), 2 % SDS, 0.2 mM Dithiothreitol, 3.6 M Urea, and 15 % (v/v) Glycerol. Monitoring of protein transfer onto membranes during Western blots. Monitoring of protein migration during SDS-PAGE. Ready-to-use: supplied in a loading buffer for direct loading on gelsĮasy to identify: includes the ~25, ~75 kDa reference bands coupled with a green and a red dye The ladder is supplied in gel loading buffer and is ready to use.īroad range: 10-245 kDa (Tris-glycine-SDS running buffer) The BLUeye Prestained Protein Ladder is designed for monitoring protein separation during, verification of Western transfer efficiency on membranes (PVDF, nylon, or nitrocellulose) and for approximating the size of proteins. ![]() Proteins are covalently coupled with a blue chromophore except for two reference bands (one green and one red band at 25 kDa and 75 kDa respectively) when separated on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with Tris-glycine-SDS running buffer. The BLUeye Prestained Protein Ladder is a three-color protein standard with 12 prestained proteins covering a wide range molecular weights from 10 to 245 kilodalton (kDa). ![]()
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