seeblue protein ladder mbd2 western blot

We identified the following criteria for the ladder proteins: We designed our protein ladder to be comprehensive as well as simple and inexpensive to prepare. We have also constructed polycistronic expression vectors to coexpress either the set of 10, 30, 50, 100 kD proteins or the set of 20, 40, 60, 80 kD proteins, enabling the efficient production of a 10–100 kD protein ladder with just two expression experiments. The ladder proteins can be detected by Coomassie Blue staining of SDS-PAGE gels and on Western blots through engineered IgG binding domains. coli and purified by metal affinity chromatography with minimal equipment. These proteins can be expressed at very high levels in E. We believe there is a similar need for inexpensive protein molecular weight markers, and we have therefore created bacterial expression vectors to express ladder proteins that range from 10 to 100 kD. We have previously described two plasmids that can be used to prepare both 100 bp and 1000 bp DNA molecular weight markers efficiently and inexpensively 6. An alternate approach allows prestained markers to be detected in Western blots using monoclonal antibodies generated against the Remazol dyes present in prestained proteins (these monoclonal antibodies are now commercially available from Diagenode) 5. To avoid this inelegant approach, protein ladders have been created to contain peroxidase activity (for detection by peroxidase-mediated chemiluminescence) 2, a medley of 14 affinity tags (to bind anti-tag antibodies) 3, or fragments of or intact IgG binding domains (for detection by peroxide-linked second antibodies) 4. The Western blot X-ray film (if using chemiluminescence) can be overlaid onto the blotted membrane and the positions of the protein ladder bands marked by pen on the X-ray film.

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. We have created the Penn State Protein Ladder system to produce protein molecular weight markers easily and inexpensively (less than a penny a lane).

Seeblue protein ladder mbd2 western blot