 Gel Electrophoresis  Gel staining  Transfer  Immunoblotting  Optimization

SDS Protein

In aqueous solutions, SDS polarizes releasing Na + and retaining a negative charge on the sulfate head So much SDS binds to proteins that the negative charge on the SDS drowns out any net charge on protein side chains In the presence of SDS all proteins have uniform shape and charge per unit length SDS non-polar chains arrange themselves on proteins and destroy secondary tertiary and quaternary structure.

Acrylamide  Polyacrilamide is a polymer made of acrylamide (C 3 H 5 NO) and bis-acrilamide (N,N’-methylene-bis- acrylamide C 7 H 10 N 2 O 2 ) O CH CH 2 NH 2 C O CHCH 2 NH 2 C CH 2 bis-Acrylamide O CHCH 2 NH 2 C Acrylamide

 Acrylamide polymerizes in the presence of free radicals typically supplied by ammonium persulfate O CHCH 2 NH 2 C O CHCH 2 NH 2 C SO 4 -.

1. Acrylamide polymerizes in the presence of free radicals typically supplied by ammonium persulfate SO 4 -. O CHCH 2 NH 2 C O CHCH 2 NH 2 C O CHCH 2 C O CHCH 2 NH 2 C 2.TMED (N,N,N’,N’-tetramethylethylenediamine) serves as a catalyst in the reaction

 bis-Acrylamide polymerizes along with acrylamide forming cross-links between acrylamide chains O CHCH 2 NH 2 C O CHCH 2 NH 2 C O CHCH 2 NH 2 C O CHCH 2 C O CHCH 2 NH 2 C O CHCH 2 NH 2 C bis-Acrylamide O CH CH 2 NH 2 C O CHCH 2 NH 2 C CH 2

 bis-Acrylamide polymerizes along with acrylamide forming cross-links between acrylamide chains

 Pore size in gels can be varied by varying the ratio of acrylamide to bis-acrylamide. Protein separations typically use a 29:1 or 37.5:1 acrylamide to bis ratio Lots of bis-acrylamide Little bis-acrylamide

1 2 3 Addition of SDS Protein becomes rod-shaped with uniform charge distribution

 Commassie Blue  Silver staining

 Two major factors affect the efficiency of protein blotting:  (1) the elution efficiency of a protein out of a gel matrix.  (2) The efficiency of binding by the membrane.

 In a general way, the lower the percentage of acrylamide and cross-linker, the easier the transfer will be.  Transfer becomes faster and more complete with the use of thinner gels.  Disruption of the gel matrix to partial proteolytic digestion of the high Mw proteins prior to the transfer.

 Nitrocellulose, polyvinylidene difluoride (PVDF), activated paper or activated nylon have all been used successfully to bind transferred proteins.  PVDF membranes offer the advantages of high protein binding capacity, physical strength and chemical stability.

 Small proteins tend to move through nitrocellulose membranes and only a small fraction of the total amount actually binds.  In addition, while nitrocellulose membranes cannot be stained with Coomassie Brilliant Blue (CBB), PVDF membranes are amenable to staining with CBB.

 Transfer of proteins from native or SDS-PAGE gels to nitrocellulose or PVDF membranes has been achieved in three different ways:  (1) simple diffusion  (2) vacuum-assisted solvent flow  (3) ‘‘Western’’ blotting or electrophoretic elution

 This protocol had not gained widespread acceptance owing to the fact that there was no quantitative transfer of protein.

 An alternative to diffusion blotting and electroblotting.  When using low-molecular-weight proteins, the authors suggest using nitrocellulose membrane with a small pore size (0.2 or 0.1 µm).

 Electroblotting has been and is the most commonly used method to transfer proteins from a gel to a membrane. The principal advantages are the speed and the completeness of transfer compared to diffusion or vacuum blotting.

 (1) complete immersion of a gel-membrane sandwich in a buffer (wet transfer) or by  (2) placing the gel-membrane sandwich between absorbent paper soaked in transfer buffer (semi-dry transfer).

 There are a number of advantages to this procedure over the conventional upright protocol. Several gels can be blotted simultaneously; electrodes can be cheap carbon blocks; less power is required for transfer (and therefore a simpler power pack).

 Under native PAGE conditions, proteins retain their higher-order structure and often retain their biological activity.  SDS and dithiothreitol (DTT) or β mercaptoethanol ( β ME) are omitted from the standard SDS protocol.  The sample buffer is devoid of SDS or DTT/ β ME and the samples are not heated. The gel and tank buffer solutions are prepared without SDS.

 Two methods are commonly used for detecting  proteins, after the addition of primary antibody to protein blots that have been blocked: (1) radioactive and  (2) enzyme-linked reagents.

 Detection with the use of enzyme-labeled antibodies had been the most popular method until the advent of the chemiluminescence method of detection.  Protein A bound to particles of gold has been used for detecting immune complexes on Western blots in the so-called ‘‘Golden blot.’’  Detection of proteins using Biotin–streptavidin system.