Name: protein size separation by sds-page
Uploaded: 2023-04-30T11:58:23.000Z
Duration: 1 min 27 s
Channel: JoVE

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis or SDS-PAGE is a molecular weight-based technique that separates mixtures of proteins based on their size.
Here, sample proteins are treated with &#946;-mercaptoethanol and SDS.
&#946;-mercaptoethanol is a reducing agent that breaks the disulfide bonds.
SDS, a negatively charged detergent, binds at the hydrophobic regions to unfold and denature the proteins and adds a uniform negative charge.
The samples are then loaded onto a polyacrylamide gel made of crosslinked acrylamide monomers.
The top stacking gel has large pores due to low acrylamide concentration.
On voltage application, the protein samples stack like coins to enter the lower resolving gel simultaneously.
The resolving gel has smaller pores due to a higher acrylamide concentration. It acts as a molecular sieve, allowing smaller proteins to run faster, thus separating the proteins based on their sizes.
These separated protein bands are commonly visualized using coomassie blue staining. The molecular weight of each band can be determined using reference marker proteins.

sds-page

SDS-PAGE

Gel electrophoresis is a method that separates biological macromolecules like nucleic acids or proteins by forcing them to pass through a gel matrix under an electric field.
A variation of gel electrophoresis, termed&#160; polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins based on their net charges and size, as seen in native PAGE. Alternatively, proteins can be denatured and coated with a negatively charged detergent called sodium dodecyl sulfate (SDS), masking the native charges and allowing separation based on size only, as seen in SDS-PAGE. Polypeptide chains can migrate in polyacrylamide gel even when coated with SDS. So, with this observation, Ulrich K. Laemmli developed the technique of SDS-PAGE in the year 1970.
In SDS-PAGE, the polyacrylamide gels are formed by the polymerization of acrylamide monomers that are transparent, chemically and biologically inert, and uncharged. These gels&#160; have a controllable pore size determined during gel preparation, where the concentration of acrylamide and bisacrylamide, the cross-linking agent, regulate the gel&#8217;s pore size. Acrylamide monomers thus polymerize to form polyacrylamide, wherein ammonium persulfate catalyzes the polymerization reaction. SDS provides a uniform charge to mass ratio for all proteins. Usually, a gram of protein is covered by 1.4 gm of SDS allowing size-driven protein separation.
The sample preparation buffer for SDS-PAGE, besides SDS and &#946;-mercaptoethanol, contains glycerol and bromophenol blue. The density of glycerol helps the sample reach the bottom of the stacking well, preventing it from flowing out from the well into the buffer. Bromophenol blue functions as the tracking dye and indicates the proteins&#8217; progress in the gel.
Optimum pH for each buffer solution is crucial as it determines ion concentration in the buffer required for protein movement under voltage application. The ionic strength and pH of the buffer used for running the gel (pH 8.3) is different from the buffers used to create&#160; the stacking gel (pH 6.8) and the resolving gel (pH 8.3). This pH difference between the stacking and resolving gel ensures that the low ionic strength of the stacking gel offers high electrical resistance, allowing the proteins to stack and then separate once they enter the resolving gel.
SDS-PAGE has varied applications, including the estimation of protein size, purity, and even peptide mapping. The major limitation of this technique is that it cannot obtain information about the enzyme activity, cofactor, and protein binding interactions. It is challenging to analyze highly acidic or basic proteins using SDS-PAGE.

Gel electrophoresis is a method that separates biological macromolecules like nucleic acids or proteins by forcing them to pass through a gel matrix under ...

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protein size separation by sds-page

SDS-PAGE separates proteins by size using a polyacrylamide gel and an electric field. It is a type of gel electrophoresis, which moves biological macromolecules through a gel matrix. In PAGE, proteins pass through polyacrylamide gel and can be separated by size, charge, or both, depending on the method used.

In native PAGE, intact proteins keep their natural charges and are separated by both net charge and size. In SDS-PAGE, proteins are first denatured and coated with sodium dodecyl sulfate, or SDS. SDS is a negatively charged detergent that masks the native charges and gives proteins a similar charge-to-mass ratio. This lets proteins move through the gel based mainly on size. The method was developed by Ulrich K. Laemmli in 1970.

The polyacrylamide gel is made by polymerizing acrylamide monomers. These monomers are transparent, chemically and biologically inert, and uncharged. Bisacrylamide acts as the cross-linking agent, and its amount helps control pore size. Ammonium persulfate catalyzes the polymerization reaction. The pore size of the gel can therefore be adjusted during preparation.

The sample buffer for SDS-PAGE contains SDS, beta-mercaptoethanol, glycerol, and bromophenol blue. Glycerol helps the sample sink into the bottom of the stacking well instead of spreading into the buffer. Bromophenol blue is the tracking dye, so it shows how far the proteins have moved in the gel. In practice, about 1.4 g of SDS coats 1 g of protein, which supports size-based separation.

Buffer pH is important for protein movement under voltage. The running buffer is usually at pH 8.3. The stacking gel buffer is at pH 6.8, and the resolving gel buffer is at pH 8.3. The low ionic strength of the stacking gel creates high electrical resistance, so proteins collect in a tight band before they separate in the resolving gel.

SDS-PAGE is used to estimate protein size, check purity, and perform peptide mapping. It is useful when proteins need to be compared by size alone. However, it does not show enzyme activity, cofactor use, or protein binding interactions. It can also be difficult to analyze highly acidic or highly basic proteins with this method.

Protein Size Separation by SDS-PAGE

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key terms and concepts

Learn how SDS-PAGE separates proteins by size using polyacrylamide gel, SDS, and buffer pH differences to estimate protein size and purity.

Protein Size Separation by SDS-PAGE | JoVE K12

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Protein Size Separation by SDS-PAGE

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