For the molecular weight estimation, SDS-PAGE is used, as sodium dodecyl (SDS) is an anionic detergent and disrupts the secondary, tertiary, quaternary structures of protein. A reducing agent marcaptoethanol (BME) is also used in this SDS-PAGE, to reduce the disulfide bonding of proteins which is in a polypeptide chain form ( due to SDS disruption). While native PAGE is used to know the aggregation state of protein. No disrupting and reducing agents such as SDS, BME are used in native PAGE.

This question has been asked before - see the comprehensive answers under the question "Can anyone detail the differences between Native PAGE and SDS-PAGE?" from April 25, 2013.

Dear Stephanie Galindo

Western blot is preferred with SDS-PAGE instead of native PAGE for a few reasons as following:

The role of SDS in SDS-PAGE is to coat the hydrophobic region of the protein with its negative charge and overcome the overall positive charge of the protein so that the protein can migrate towards the positive electrode. Therefore, the proteins in the sample can be resolved according to their molecular mass, whereas in native PAGE, proteins are separated according to their charge to molecular mass ratio.

SDS is an anionic denaturing agent which denatures the secondary, tertiary and quaternary (if present) structure of the proteins. SDS-PAGE includes reducing agents such as β-mercaptoethanol (BME), dithiothreitol (DTT) which cleaves the disulphide bridges present in the protein, results in destabilizing the native secondary structure, thereby completely unfolding the protein. In native PAGE, neither denaturing agent nor reducing agent is used, thus proteins retain their native conformations.

The fundamental principle of western blot is that antigen-binding region (paratope) of primary antibody (1°Ab) recognizes and binds specifically to epitope of antigen (here the desired protein in sample). The epitope is mostly unhampered following the treatment of SDS and reducing agents because the epitope is commonly a linear amino acid sequence (or short peptide stretch). Although the availability of the epitope should be validated each time as denaturation of the protein (here antigen) may affect the antibody binding affinity. Native PAGE is only employed where the epitope is a discontinuous stretch of amino acid (called conformational epitope), so that the primary antibody can only recognize the native protein epitope, e.g. for plasma membrane protein.

I hope this will help you.

Best regards.

To put it simply, SDS-PAGE separates protein based exclusively on mass. Protein migration in Native PAGE is based on charge to mass ratio, meaning the lower band you can visualize may not represent a low molecular weight protein but protein that is highly negative charge.

Proteins can be transferred from a gel to a blotting membrane if there is SDS in the gel . In the case of native PAGE there is no SDS and there will be less effective transfer of protein.

If you fix and stain a gel with a dye ( eg. Coomassie Blue) and then wash the stained gel with water and then try to blot the proteins there will be little transfer to the blot . However if you wash the stained gel with water and then with a buffer containing SDS (for instance the electrode buffer used in the Laemmli method of SDS-PAGE which has 0.1% SDS) and then do the blotting the proteins will transfer. However, if you wash the gel in SDS for too long you will loose protein from the gel before the blot procedure.

This method has been described :

Jackson, P. & Thompson R.J.

Electrophoresis 1984, Vol 5, p35-42.