Last Updated on
July 18, 2023
By
Excedr
“How do you determine the actual size or molecular weight of a protein when separated in a gel?”
The answer is simple—using protein standards!
A protein standard is a mixture of known, well-characterized, natural or recombinant proteins of different sizes. They are loaded in a gel alongside experimental protein samples as a reference for protein sizes (in kilodalton or kDa). They are also known as molecular weight markers, protein markers, or molecular weight standards.
The protein markers are widely used in monitoring the electrophoretic separation process and the concentration and molecular weight determination of the proteins. This separation of proteins is performed using protein electrophoresis techniques, such as sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE).
With a range of protein standards available in the market, such as prestained protein standards spectra multicolor broad range standards, and unstained proteins standards, choose standards that provide:
During electrophoresis, prestained standards enable the direct visualization of protein migration and aid in assessing their transfer to membranes.
While prestained standards can be utilized for size estimation, the most accurate size determination is achieved by using unstained protein standards.
In this article, we will cover how protein standards help in blot detection by creating reference bands and what are the workflows that involve this reagent for many life sciences and molecular biology research studies.
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Protein ladders help in the analysis of experimental proteins by creating reference bands within the gel through protein gel electrophoresis. They consist of a mixture of proteins with known molecular weight and other characteristics.
Protein ladders are introduced to gels alongside protein samples, the standards migrate through the gel in response to an electrical current. Once the electrophoresis run is finished, the proteins in the standard mixture will separate and form distinct bands along the gel, due to their different sizes.
By comparing the migration positions of the protein standards with the unknown protein samples, scientists can estimate the sizes or molecular weights of the proteins in the samples. This helps in characterizing and analyzing the protein samples based on their relative sizes and allows for a better understanding of their behavior in different experimental conditions.
The two types of widely used protein ladders include:
Natural prestained proteins can exhibit variations in bandwidth due to differences in the binding sites of the dye, while recombinant proteins tend to produce sharper and more defined bands. Therefore, recombinant protein ladders are generally preferred over natural protein ladders for accurate molecular weight estimation.
Engineered recombinant protein ladders are designed to generate distinct and well-defined bands with uniform molecular weights, offering enhanced precision. These protein ladders can be stained with various colors or labeled with affinity tags, enabling effortless identification and facilitating their detection in western blot experiments.
Prestained markers retain visibility on the membrane post-transfer, allowing active monitoring of transfer efficiency and antigen localization in repetitive screening assays.
However, the proteins become visible only after staining with a Coomassie Brilliant Blue R250 stain or any non-specific protein stain.
Unstained protein ladders provide more precise protein sizing since the dyes in prestained ladders can subtly alter the appearance of proteins on the gel.
Figure: Types of Bio-Rad’s protein standards for use in western blot assay.
Protein standards are widely used in gel electrophoresis to determine the size of the experimental proteins. It is required for downstream applications, such as western blotting, protein sequencing, and mass spectrometry.
Western blotting is a frequently used technique in molecular biology labs to analyze various proteins and peptides using primary and secondary antibodies (conjugated with enzymes). Additionally, it’s also used to verify protein purification after cloning. The standard provides reference bands to match with the unknown proteins for the determination of their size.
There are also Western blotting protein standards that are used for both chemiluminescent immunodetection or fluorescent visualization and colorimetric analysis on western blots. Some companies supply protein standards with ten prestained, Strep-tagged recombinant proteins. It enables researchers for colorimetric or chemiluminescent western blot detection, with StrepTactin-HRP or StrepTactin-AP Conjugate, and fluorescent visualization on gels.
A range of protein ladders (molecular weight markers) is available in the market for use in protein electrophoresis and western blotting applications. The workflows are extensively used in various industries for research or medical interventions.
Protein standards are used for various purposes, including quality control, method validation, and assay development. They are used to establish reference points and calibration curves for protein assays, such as enzyme-linked immunosorbent assays (ELISAs) or protein quantification methods.
They help ensure accurate and reliable measurements of protein concentrations in pharmaceutical samples.
Protein standards are valuable tools for researchers in the biotechnology industry. They have the following uses in the lab:
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Protein standards are a mixture of well-characterized proteins with known molecular weight and other characteristics. These well-characterized reference proteins enable researchers to monitor electrophoretic separation, estimate protein size and concentration, verify protein transfer onto membranes, and ensure accurate molecular weight determination.
They play a crucial role in various scientific applications, including protein electrophoresis and Western blotting. To perform such high-throughput downstream applications, require you to work with quality reagents (refer to FAQs, safety datasheet, and other documents that come with chemicals to learn more about them) and advanced equipment. And, the expense associated with buying the equipment can be heavy on your pocket.
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