Why study proteins? Proteins are fascinating molecular devices. They play important roles in the life processes. There are structural proteins , catalytic proteins, transport and storage proteins, regulatory proteins, proteins of the immune system and the immunoglobulin superfamily, cell-cell recognition and signaling proteins.

Estimation of Proteins

Assay of Protein Purity, Molecular Weight, and Subunit Structure

SDS Polyacrylamide Gel Electrophoresis of Proteins SDS-PAGE is the most widely used method for qualitatively analyzing protein mixtures. It is particularly useful for monitoring protein purification, and because the method is based on the separation of proteins according to size, the method can also be used to determine the relative molecular mass of proteins.

Formation of Polyacrylamide Gels: Crosslinked polyacrylamide gels are formed from the polymerization of acrylamide monomer in the presence of N,N'-methylene-bis-acrylamide . Bisacrylamide is essentially two acrylamide molecules linked by a methylene group and is used as a crosslinking agent. Acrylamide monomer is polymerized in a head-to-tail fashion into long chains, and occasionally a bis-acrylamide molecule is built into the growing chain, thus introducing a second site for chain extension. Proceeding in this way, a crosslinked matrix of fairly well-defined structure is formed.

Polymerization of Acrylamide

The polymerization of acrylamide is an example of free-radical catalysis, and is initiated by the addition of ammonium persulfate and the base N,N,N',N'-tetramethylenediamine (TEMED). TEMED catalyzes the decomposition of the persulfate ion to give a free radical (a molecule with an unpaired electrons.

The Use of Stacking Gels The purpose of the stacking gel is to concentrate the protein sample into a sharp band before it enters the mail separating gel, thus giving sharper protein bands in the separating gel. This modification allows relatively larger sample volumes to be applied to the gel without any loss of resolution. The stacking gel has a very large pore size (4% acrylamide) which allows the proteins to move freely and concentrate, or stack under the effect of the electric field. Sample concentration is produced by isotachophoresis (the band-sharpening effect) of the sample in the stacking gel. The isotachophoresis relies on the fact that the negatively charged glycinate ions (in the reservoir buffer) have a lower electrophoretic mobility than the protein-SDS complexes, which in turn have lower mobility than the chloride ions if they are in a region of higher field strength. Field strength is inversely proportional to conductivity, which is proportional to concentration. The result is that the three species of interest adjust their concentrations so that [chloride ion]>[protein-SDS]>[glycinate]. There are only a small quantity of protein-SDS complexes, so they concentrate in a very tight band between the glycinate and chloride ions boundaries. Once the glycinate reaches the separating gel, it becomes more fully ionized in the higher pH environment and its mobility increases. (the pH of the stacking gel is 6.8 and that of the separating gel is 8.8). Thus the interface between glycinate and chloride ions leaves behind the protein-SDS complexes, which are left to electrophorese at their own rates.

SDS-PAGE Samples to be run on SDS-PAGE are first boiled in sample buffer containing β-mercaptoethanol or DTT (Dithiothreitol) and SDS. The mercaptoethanol reduces any disulfide bridges present that are holding together the protein tertiary structure. SDS is an anionic detergent and binds strongly to, and denatures , the proteins. Each protein in the mixture is therefore fully denatured by this treatment and opens up into a rod-shaped structure with a series of negatively charged SDS molecules along the polypeptide chain. On average, one SDS molecule binds for every two amino acid residues. The sample buffer also contains an ionizable tracking dye usually bromophenol blue that allows the elctrophoretic run to be monitored. Sucrose or glycerol which gives the sample solution density. Thus allowing the sample to settle easily through the electrophoresis buffer.

Silver-Stained SDS-PAGE

Standard Curve for the Estimation of Protein MWs by SDS-PAGE

Gradient SDS-PAGE The use of PAGE that have a gradient of increasing acrylamide concentration (and hence decreasing pore size) can sometimes have advantages over fixed-concentration acrylamide gels. There are two main advantages of gradient gels over linear gels. A much greater range of protein Mr values can be separated than on a fixed percentage gels. In a complex mixture, very low-mol-wt proteins travel freely through the gel to begin with, and start to resolve when they reach the smaller pore size toward the lower part of the gel. Much larger proteins can still enter the gel but start to separate immediately owing to the sieving effect of the gel. Proteins with very similar Mr values may be resolved that can not be resolved in fixed percentage gels. As each protein moves through the gel, the pore size become smaller until the protein reaches its pore size limit. The pore size in the gel is now too small to allow passage of the protein, and the protein sample stacks up at this point as a sharp band. A similar-sized protein, but with slightly lower Mr, will be able to travel a little further through the gel before reaching its pore size limit, at which point it will form a sharp band. These two proteins, of slightly different Mr values, therefore separate as two, close, sharp bands.

Silver Stained SDS-PAGE

Gradient forming Apparatus

Gelatin-SDS-PAGE Zymography Zymography is a method for the detection of a specific enzyme among the bands separated by electrophoresis.

Isoelectric Focusing (IEF) A protein has charged groups of both polarities and therefore has an isoelectric point, pI, at which it is immobile in an electric field. If a mixture of proteins is electrophoresed through a solution or gel that has a stable pH gradient in which the pH smoothly increases from anode to cathode, each protein will migrate to the position in the pH gradient corresponding to its pI. If proteins are distributed throughout a solution in a pH gradient, then upon application of an electric potential across the gradient, with the anode at the low pH end, molecules in the low pH zone (which will be positively charged) will migrate to the cathode. Conversely, molecules in the high pH zone will be negatively charged and will consequently migrate, through zones of decreasing pH, towards the anode. When each protein reaches a position where the pH is equal to its pI, it will lose all of its charge and its migration will cease.

2-D Electrophoresis In 2-D electrophoresis, proteins are separated in the first dimension, according to their isoelectric point by IEF, and then the separated proteins are subjected to SDS-PAGE in the perpendicular direction. Two-dimensional PAGE is a valuable tool for proteomics, a field of study that involves cataloguing all of a cell’s exposed proteins with emphasis on their characterization and functional activities.

Western Blots Immunoblotting (Alternatively, Western blotting) is a method to detect a specific protein in a given sample of tissue homogenate or extract. It uses SDS-PAGE to separate polypeptides. Then, the polypeptides are electrophoretically transferred to a membrane ( typically nitrocellulose or PVDF). The transferred proteins are bound to the surface of the membrane where they are probed using antibodies specific to the target protein.

Immunoblots

Protein-Protein Interaction The interactions between proteins are important for many biological functions. Signals from the exterior of a cell are mediated to the inside of that cell by protein-protein interactions of the signalling molecules. This process, called signal transduction, plays an important role in many biological processes and in many diseases. Proteins might interact for a ling time to form part of a protein complex, a protein may be carrying another protein ( for example, from cytoplasm to nucleus or vice versa in the case of the nuclear pore importins). A protein may interact briefly with another protein for the modification ( for example, a protein kinase will add a phosphate to a target protein. This modification of proteins can itself change protein-protein interaction. For example, some proteins with SH2 domains only bind to other proteins when they are phosphorylated on the amino acid tyrosine. In conclusion, protein-protein interactions are of central importance for virtually every process in a living cell. Information about these interactions improves our understanding of diseases and can provide the basis for new therapeutic approaches.

Methods to investigate protein-protein interactions Co-immunoprecipitation is considered to be good standard assay for protein-protein interactions, especially when it is performed with endogeneous (not over expressed and not tagged) proteins. The protein of interest is isolated with a specific antibody. Interaction proteins which adhere to this protein are subsequently identified by Western blotting. Interactions detected by this method are considered to be real. However, this method can only verify interactions between suspected interaction partners. Thus, it is not a screening approach.

Identification of Ras-associated proteins by coimmunoprecipitation analysis

Coimmunoprecipitation Analysis of Cauda Epididymal Fluid

Continued.. Fluorescence resonance energy transfer (FRET) is a common technique when observing the interactions of only two different proteins. Pull down assays are a common variation of IP and are used identically. A pull-down assay is distinct from IP in that it requires ligands other than an antibody to capture the protein complex. The yeast two-hybrid screen reveals the interaction between artificial fusion proteins inside the nucleus of yeast. This approach can identify partners of a protein in an unbiased manner. However, the method has a high false-positive rate which makes it necessary to verify the identified interactions by co-IP. Chemical cross linking followed by High mass MALDI mass spectrometry can be used to analyze intact protein interactions in place before trying to isolate/identify interacting proteins. This method detects interactions among non-tagged proteins.