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What is a Protein Ladder or Protein Marker?

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Protein ladders are used in experiments to estimate the size of proteins of interest and are resolved using gel electrophoresis. They are also known as protein markers, protein standards, or protein molecular weight markers.

Protein ladders contain a mixture of highly purified proteins of defined or known molecular weights. They act as a point of reference for proteins during electrophoretic transfers in western blots.  

They have a broad range of applications in life sciences and biochemistry labs for protein analysis, such as understanding the functional roles and mechanisms of proteins and protein discovery.

This article introduces the working mechanism of protein ladders and several different types that are extensively used in biological and chemical assays.

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How Do Protein Ladders Work?

A protein ladder is used to estimate the approximate size of protein molecules during gel electrophoresis. 

The mixture of purified proteins that makes up the protein ladder moves through the gel and is sequentially arranged according to their molecular weight, i.e., from largest molecular weight (closest to the sample loading well) to lowest molecular weight (farthest from the sample loading well). 

This is because: 

  • The smaller-sized proteins can easily move through the small pores of the gel matrix and move farthest from the sample loading well, i.e., towards the positive electrode. 
  • The movement of larger-sized proteins through the small pores of the matrix is hindered, causing them to remain closer to the sample loading well, i.e., closer to the negative electrode.

Figure: An illustration showing the sample loading lane and direction of movement of proteins during protein gel electrophoresis.

The movement of proteins in the gel matrix also depends on the SDS-PAGE buffer systems (whether it’s a Tris-Glycine buffer, Bis-Tris MOPS buffer, or Bis-Tris MES buffer). The protein ladders in the gel are visible in the form of bands and serve as reference bands to denote the size of sample proteins.

Figure: A picture of protein electrophoresis with protein ladders in the first column of the immunoblot.

Common Types of Protein Ladder

Prestained Protein Ladders

Prestained protein ladders, as the name suggests, have protein mixtures prestained with colors. 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). These bands make it easier to monitor the protein migration patterns and calculate the molecular weights of proteins.

Prestained protein ladders typically come in two types: 

  • Prestained naturally-occurring protein ladders: These produce broad bands when stained with one or more colors and show variability in the amount and location of dyes binding to each protein. Researchers typically do not prefer these because they tend to produce indistinct broad bands.
  • Prestained recombinant protein ladders: These are highly preferred ladders for research use. They are engineered so that they produce sharp, evenly-spaced proteins prestained with different colors, making it easier and simpler to estimate the approximate molecular weight of proteins.

Most commercial prestained protein ladders are available as three-color protein standards with 10-12 prestained proteins. They are supplied in a loading buffer for their direct loading in gel lanes. 

The storage buffer used for these ladders consists of a mixture of reagents like Tris citrate/phosphate, urea, sodium lauryl sulfate, DTT, EDTA, and 15% glycerol in different concentrations.

Figure: An image of pre-stained protein ladders with different colors.

Prestained protein ladders have applications in:

  • SDS-Polyacrylamide gel electrophoresis (or SDS-PAGE) to monitor protein separation.
  • Western blot transfers to test the transfer efficiency of proteins on PVDF, nylon, or nitrocellulose membranes from protein gels.
  • Calculating the apparent molecular weight of proteins from the image of the membrane blot with a ladder by comparing it to the image obtained after staining proteins with antibodies.

Unstained Protein Ladders

Unstained protein ladders are a mixture of easy-to-purify proteins that are not stained with any color stains. They allow the formation of identifiable sharp bands in gels, leading to the accurate estimation of molecular weights of proteins

The bands in the gel are most commonly visualized using Coomassie Brilliant Blue R-250 stains. On the membrane blot, they are visualized using Ponceau S or any other similar stains.

Unstained protein ladders allow the accurate estimation of proteins because, unlike prestained protein ladders, their movement in the gel matrix is not hindered by the binding of dyes.

Figure: An image of unstained protein ladders. The bands are stained with Coomassie blue for visualization. 

Western Blot Protein Ladders

Western blot protein ladders are a mixture of pre-stained, StrepTactin-HRP Conjugate proteins (or Strep-tagged recombinant proteins). It enables fluorescent visualization of protein gels and chemiluminescent or colorimetric immunodetection of western blots.

Figure: Visualization of western blots using different techniques after the complete transfer of western blot protein ladders on the membrane.

Fluorescent Protein Ladders

Fluorescent protein ladders are used in SDS-PAGE gels or polyacrylamide gel electrophoresis (involving tris-glycine buffer). They contain a mixture of proteins that are pre-stained with fluorescent dyes. The bands fluoresce when the light of a specific wavelength falls on them. 

These ladders are popular among researchers because they only have a slight background interference, sensitivity, and an optimal linear dynamic range.

The light intensity of bands observed using fluorescent protein ladders is proportional to the concentration of proteins, i.e., the more the concentration of protein, the more light intensity of the bands. This makes it easier for researchers to visualize and quantify the protein bands.

Figure: An image showing gels and blots with fluorescent protein ladders.

*Note: It’s always recommended to refer to a manufacturer’s or company’s datasheet to learn more about the research uses or any detailed information on the commercially available protein ladders.

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Protein ladders are a mixture of proteins that act as points of reference during protein gel electrophoresis. They move in the gel matrix based on their sizes (from largest protein to smallest protein), in the direction of negative to the positive electrode.

Protein ladders are available in different types. Some of the most common and widely used include prestained, unstained, western blot, and fluorescent protein ladders. These are extensively used in gel electrophoresis and western blot assays to monitor protein movement and quantify bands.

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