Protein Standards & Ladders - UK (2024)

FAQs

Are all protein ladders the same? ›

Prestained Protein Ladders

They can have either all proteins prestained with one color, two or more proteins prestained in different colors, or all proteins prestained with different colors. Prestained protein ladders have three highly-intensified colored reference bands of 25 kDa, 50 kDa, and 75 kDa (kilodaltons).

Protein ladders or molecular weight markers are among the most commonly used reagents in biochemistry experiments. They provide molecular weight standards to estimate the size of proteins separated by gel electrophoresis like SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).

Transfer the desired amount of the Prestained Protein Ladder to a separate tube. For blotting: use 5 µl for mini-gels and 10 µl for full length gels. For visualizing during electrophoresis: use 10-15 µl for mini-gels and 20-30 µl for full length gels.

Protein ladders, also known as protein markers or protein standards, are used to help estimate the size of proteins separated during electrophoresis. They serve as points of reference because they contain mixtures of highly purified proteins with known molecular weights and characteristics.

b) Type I – These ladders have a duty rating of 250 pounds. Type I ladders are manufactured for heavy-duty use. c) Type II – These ladders have a duty rating of 225 pounds. Type II ladders are approved for medium-duty use.

A marker or ladder is a set of DNA fragments and the base pair length of each fragment is known. It is considered a standard because it can be used as a tool from which to measure the lengths of your unknown DNA fragments.

A DNA ladder is a solution of DNA molecules of different lengths used in agarose or acrylamide gel electrophoresis. It is applied as a reference to estimate the size of unknown DNA molecules that were separated based on their mobility in an electrical field through the gel.

However, an unstained protein ladder can only be visualized following staining with Coomassie or a similar non-specific protein stain. Unstained protein ladders are more accurate for sizing proteins, as the dyes used in prestained ladders can slightly distort the apparent size of the protein ladder proteins on the gel.

While making gel solution, TEMED has to be added last since it will immediately start to react with APS, and catalyze the polymerization of acrylamide and bisacrylamide. As a consequence, the following mixing and casting steps have to be completed as quickly as possible.

AZURE SDS -PAGE TIP #4: Load the appropriate amount of protein sample. Loading an excess of protein per well can cause the proteins to aggregate during electrophoresis, preventing them from separating by size and causing them to run together. This can result in clustered bands that cannot be individually defined.

How much protein is too much for western blot? ›

To obtain linear signals with the majority of western blots, we recommend loading smaller amounts of protein sample between 1 and 10 μg per well. To avoid under- or overloading samples, determine the protein concentration of each sample prior to electrophoresis with a compatible protein assay.

Load the following volumes of the ladder on an SDS-polyacrylamide gel: – 5 μL per well for mini gel, – 10 μL per well for large gel. Use the same volumes for Western blotting. The loading volumes listed above are recommended for gels with a thickness of 0.75-1.0 mm.

The PageRuler™ Unstained Broad RangeProtein Ladder is a mixture of eleven recombinant, highly purified proteins ranging from 5 kDa to 250 kDa. For easy reference, the 100 kDa, 50 kDa and 20 kDa protein bands have greater intensity than the other proteins in the ladder.

The smallest bands are at the bottom of the gel (smaller DNA fragments run through the gel more quickly than larger fragments), the larger bands are at the top. To estimate the size of your PCR amplicon, you can plot an imaginary line to the right or left of your amplicon and see how far it is up the ladder scale.

The most important factor in choosing a DNA ladder is the expected size of the DNA bands in your experiment. You want to make sure that the range of the DNA ladder, from its smallest to its largest fragment, includes the expected size of the DNA fragments you are testing (Figure 1).

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Figure 3-2. The structural components of a protein. A protein consists of a polypeptide backbone with attached side chains. Each type of protein differs in its sequence and number of amino acids; therefore, it is the sequence of the chemically different side chains (more...)

Not all Protein is Equal

Protein sources react differently in the body based on unique absorption rates and differing levels of both essential and non-essential amino acids. The unique amino acid composition of proteins affects how the body is able to utilize them for growth, recovery & maintenance.

We recommend using unstained protein ladders for molecular weight estimation applications as prestained ladders have a dye that is covalently bound to each protein that will result in the ladder migrating differently in different buffer systems (i.e., different gels).