In analytical biochemistry rulers and markers are crucial to interpret the result of research. Without them the segregation of DNA or proteins into fragments via gel electrophoresis or e.g. SDS-PAGE cannot be put into context. Scientist run an additional lane with a molecular weight marker. It contains several defined amounts of DNA or protein in the range of the research object. The separated bands together look like a ladder on the gel therefore the name protein or DNA ladder. By comparing the bands of the probe with the ladder it is possible to deduct the length. Although ladders are not intended for use in quantitative analysis, one can get a rough estimate of the amount of experimental probe from the relative intensity of the probe and a band in the molecular weight marker that is similar in size and the (calculated) amount of the marker band on the gel.
As proteins and nucleic acids are mostly colorless, their progress through the gel during electrophoresis cannot be easily followed. Anionic dyes of a known electrophoretic mobility are therefore usually included in the sample buffer. Being highly mobile molecules they move ahead of most proteins/ DNA. As it reaches the anodic end of the electrophoresis medium electrophoresis is stopped. Common tracking dyes are Xylene cyanol, cresol Red, Bromophenol blue, Orange G or Tartrazine. They differ slightly in mobility.
DNA Ladders & staining
To visualize DNA bands, ethidium bromide has been the predominant dye used for nucleic acid staining for decades because of its low price and generally sufficient sensitivity. However, several drawbacks of ethidium bromide have been understood, including that ethidium bromide is a mutagen/carcinogen and presents a high risk of inducing cancer. Moreover, the ultraviolet (UV) light used to illuminate EtBr-DNA compounds probably results in skin or eye damage to the user if misconducted. It’s also noted that exposure to the UV light might cause chemical modifications of the DNA samples in the gel, such as the formation of TT dimers, leading to challenges with the subsequent DNA manipulations.
For this reason, ethidium bromide alternative DNA gel stains, such as SYBR®, GelRed™ and GelGreen™ or Novel Green dyes, have become commercially available in recent years. Although these alternative nucleic acid staining dyes have reduced mutagenicity, they sometimes have to sacrifice other aspects of the dyes. The DMSO stock solution, in which most of them are soluted, should be handled with particular caution as DMSO is known to facilitate the entry of organic molecules into tissues.
GelRed™ and GelGreen™ obtain reduced genotoxicity by preventing the dyes from entering living cells. They are incapable of crossing cell membranes. Furthermore, environmental safety tests showed that GelRed™ and GelGreen™ are nonhazardous and nontoxic to aquatic life.
SYBR Green I is marketed as a replacement for the potential human mutagen ethidium bromide, as both safer to work with and free from the complex waste disposal issues of ethidium.
The Novel Green (external link) provides an easy 2-step method to stain the DNA band from DNA electrophresis. This reagent ensures the DNA to be stained with a high sensitivity and good quality on the gel. The producer commends it for usage in quantitative for more applications, e.g. real-time PCR (qPCR) aswell. If Novel Green is suitable for your work, we recommend our product OneMARK 100 (ABIN2868514) we provide in our shop.
As mentioned above the correct marker choice depends on the length of DNA fragments of interest. If the fragments differ in length by a big margin, our product Lambda DNA/Hind III Marker (ABIN1540476) might be the marker of choice. Isolated DNA of Enterobacteria phage λ digested by the restriction enzyme Hind III leads to a mixture of DNA fragments with defined length covering a broad spectrum.
Protein Ladders & staining
Protein ladders are designed for a variance of applications like monitoring protein separated during SDS-polyacrylamide gel electrophoresis, verification of Western transfer efficiency on membranes (PVDF, nylon, or nitrocellulose) or approximate sizing of proteins. To make the proteins visible, a protein-specific, dye-binding or color-producing chemical reaction must be performed on the proteins within the gel. The most common ones are listed below.
Coomassie Brilliant Blue is the most popular protein stain. It is easy to handle and does not permanently chemically modify the target proteins. It is an anionic dye, which non-specifically binds to proteins. The structure of Coomassie Brilliant Blue is predominantly non-polar, and it is usually used in methanolic solution acidified with acetic acid. Proteins in the gel are fixed by acetic acid and simultaneously stained. Coomassie dye binds to basic and hydrophobic residues of proteins, changing in color from a dull reddish-brown to intense blue. The excess dye incorporated into the gel can be removed by destaining with the same solution without the dye. Classical Coomassie Brilliant Blue staining can usually detect a 25-50 ng protein band.
Concerning the coloration of the protein ladder it is more and more common to dye them in different colors for easier orientation on the gel. Our Smart Multi Color Pre-Stained Protein Standard (ABIN1536541) for example consists of six pre-stained proteins in the range of 14 to 100 kDa. The proteins of 40 kDa and 100 kDa are covalently coupled with orange dye, the protein of 14 kDa is covalently coupled with bright yellow dye.
Silver staining is the most sensitive colorimetric method for detecting total protein. The technique involves the deposition of metallic silver onto the surface of a gel at the locations of protein bands. Silver ions interact and bind with certain protein functional groups. It combines excellent sensitivity whilst using very simple and cheap equipment and chemicals In comparison with CBB staining the sensitivity increases 30-100 times. However, silver staining can be difficult in usage, as many artefacts and pitfalls exist. The extreme protein to protein variability in staining and the wide range of colors obtained for various proteins, leading in some cases to "hollow staining" with a transparent or weakly colored center of the protein zone and a more deeply colored periphery.
A number of fluorescent stains for total protein have been introduced in recent years. Newer fluorescent total-protein stains provide exceptional fluorescent staining performance with fast and easy procedures. The most useful are those whose excitation and emission maxima correspond to common filter sets and laser settings of popular fluorescence imaging instruments.
Our Protein Marker for Fluorescent Western Blotting (ABIN1536542) is designed for convenient protein band identification in fluorescent Western Blotting. It enables direct visualization of both the protein marker and users' samples on the same western blot membrane without any additional reagents.
Some products cover both colometric and fluorescence staining e.g. Chameleon™ 800 Pre-stained Protein Ladder (ABIN2737872). It is a mixture of 6 recombinant proteins (8 to 260 kDa) for multi-colored visual and 800 channel near-infrared detection. The ready-to-use ladder provides convenient, consistent sizes for SDS-PAGE applications and is ideal for monitoring electrophoretic separation, molecular weight sizing, and monitoring blot transfer efficiency.
Chemiluminescence is the production of visible light (luminescence) occurring as a result of a chemical reaction. Chemiluminescence is typically about 2 orders of magnitude more sensitive than fluorescence.