A Guide to Prestained Protein Ladders: What, Why, and How (2024)

Prestained Protein Ladders play a vital role in molecular biology and protein research. They are essential tools used to determine the molecular weight of proteins which are separated by gel electrophoresis. This article aims to guide you about prestained protein ladders, covering key aspects of what they are, why people choose them, and how to use them properly. A proper understanding of the prestained protein ladder is fundamental for successful protein analysis and interpretation of results.

A Guide to Prestained Protein Ladders: What, Why, and How (1)

What is a Prestained Protein Ladder?

A prestained protein ladder serves as a molecular weight standard in molecular biology. It allows visualization of protein bands separated by electrophoresis to gauge protein sizes accurately. A prestained protein ladder contains a mixture of purified proteins or peptides of known molecular weights. These marker proteins act as visible reference points on gels.

Purpose of Prestained Protein Ladders

A prestained protein ladder is specifically designed to aid in identifying target proteins separated by electrophoretic techniques like SDS-PAGE. It helps correlate the electrophoretic mobility of unknown protein samples to a known set of molecular weights. This allows the assignment of an approximate size or mass to proteins in a sample.

Usage in Gel Electrophoresis and Protein Analysis

During gel electrophoresis and protein analysis, prestained protein ladders are loaded into separate wells along with protein samples. As an electric current is applied, the mixture of marker proteins will migrate through the gel at rates dependent on their sizes. The presence of visible dyes bound to the proteins allows direct visualization of distinct bands corresponding to each protein size under UV light.

The Role of Visible Markers in the Ladder

It is the presence of visible dyes conjugated to marker proteins that sets prestained protein ladders apart. An unstained, standard protein ladder requires additional staining steps after electrophoresis to visualize protein bands. However, a prestained protein ladder contains already stained proteins, hence streamlining the process and allowing simultaneous development of sample and standard bands for direct comparison.

Why Do People Choose Prestained Protein Ladders?

Prestained protein ladders offer several key advantages over traditional unlabeled protein standards, which makes them very appealing to researchers seeking productive and cost-effective solutions for their protein analysis needs. Critical reasons for their increased popularity include:

Accuracy in Determining Molecular Weights of Proteins

The precise molecular weights provided by a prestained protein ladder facilitate accurate sizing of separated protein bands. Since standard proteins will migrate based on known molecular weights, their movement can be used to generate a calibration curve correlating distance travelled to log molecular weight. In this way, the size of unknown proteins can be determined to within kilodaltons.

Streamlining Protein Quantification and Identification

Being directly visible without extra staining, prestained protein ladders simplify protein analysis workflows. Researchers can immediately spot target bands, extract approximate molecular weights, and proceed to protein identification assays. Complexity and turnaround time are reduced compared to other standards that require additional development steps.

Time and Cost Savings in the Lab

The use of prestained protein ladders provides considerable time and monetary benefits in the lab. Ready-to-use prestained protein ladder mixtures bypass extra staining procedures, allowing faster experimental turnaround. Fewer reagents and consumables lower overall assay costs.

How to Use Prestained Protein Ladders

Correct usage of the prestained protein ladder is essential for obtaining accurate and reproducible results. The premixed formats of these ladders have standardized procedures for sample preparation and electrophoresis. Following the basic steps below ensures prestained markers are correctly incorporated into protein analyses:

1.Adding the Ladder to Your Gel

  • Coat a polyacrylamide or agarose gel with a sample buffer containing a prestained protein ladder.

  • Let the mixture enter the separating layer before applying a stacking gel on top.

2.Running the Gel and Visualizing Protein Bands

  • Load ladder and protein samples into separate wells.

  • Pass an electric current through the gel to separate protein/dye conjugates based on size.

  • Once electrophoresis ends, examine the gel under UV light.

3.Interpretation of Results and Molecular Weight Determination

  • Distinct colored bands representing marker proteins will be visible without further processing.

  • Measure the migration distance of each ladder band and prepare a standard curve.

  • Match unknown protein bands to standardized sizes for molecular weight determination.

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A Guide to Prestained Protein Ladders: What, Why, and How (2024)


What is the difference between unstained and prestained protein ladder? ›

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).

What is the purpose of the protein ladder? ›

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).

How do you dilute a protein ladder? ›

For silver stain applications, dilute the protein ladder approximately 1/50 in reducing sample buffer. If additional bands appear in the protein ladder, add newly prepared dithiothreitol (DTT) solution to 100mM final concentration. DTT oxidation in the storage buffer can cause the appearance of additional bands.

What is a pre stained protein ladder? ›

Prestained protein ladders

What this means is that before even loading the ladders into the gel for electrophoresis, these ladders are previously stained and ready to go. During electrophoresis, the polypeptides of the ladder show up in the gel as distinctly colored bands.

What is the purpose of the prestained standards? ›

Prestained standards allow direct visualization of the proteins' migration during electrophoresis and are useful to assess their subsequent transfer to membranes. Prestained standards can be used for size estimation, however unstained protein standards are recommended for the most accurate size determination.

How to use an unstained protein ladder? ›

Note: Dilute the ladder approximately 1/10 in reducing sample buffer for silver staining. 4. Return the unused protein ladder to -20 °C. Load 5 µL of the diluted ladder per well for a mini gel/blot and 10 µL per well for a large gel/blot.

What are prestained protein standards? ›

The Color Prestained Protein Standard is designed for observing protein separation during SDS-PAGE, verification of western transfer efficiency on membranes, and for approximating the size of proteins (1-2). The Color Prestained Protein Standard may also be used in fluorescent imaging of SDS-PAGE.

What are the different types of protein ladders? ›

Protein ladders are available in different types. Some of the most common and widely used include prestained, unstained, western blot, and fluorescent protein ladders.

What ladder to use for western blot? ›

For western blotting, use any of the SDS-PAGE ladders or those optimized for chemiluminescence detection. Whatever your needs for protein electrophoresis and western blotting, there is at least one protein ladder that meets all your requirements.

How much protein ladder to load in gel? ›

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.

Can you dilute protein powder too much? ›

Nothing will happen to your protein, but the texture and taste may change. Using too much water can result in a thin, bland shake that doesn't have very much taste.

How much ladder to use in gel? ›

For a standard electrophoresis system, we recommend loading 0.5 µg (20 µl) of the Fast DNA Ladder on the agarose gel. For a fast electrophoresis system (5 to 30 minutes separation), follow the system's manufacturer recommendations: 5 to 20 µl load. A dilution of the ladder may be required.

What is the difference between prestained and unstained protein ladder? ›

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.

Why do we need a protein ladder? ›

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.

What is the prestained protein ladder broad range? ›

Prestained Protein Ladder ab116028 is a three-color protein standard with 12 pre-stained proteins covering a wide range of molecular weights from 10 to 245 kDa.

What is protein unstained standard? ›

These unstained protein standards include three high-intensity reference bands (25, 50, and 75 kD). Precision Plus Protein Unstained Standards contain the unique Strep-tag affinity peptide, which allows detection and molecular weight determination on western blots.

How do the stained and unstained cells differ? ›

The cytoplasm in a stained cell appears more visible due to enhanced contrast from the staining process, showing details and structure, whereas in unstained cells, the cytoplasm is nearly invisible under bright-field microscopy.

Are all protein ladders the same? ›

Common Types of Protein Ladder

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).

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