Weber State University

                Department of Botany

08-Macromolecule Techniques

Recognizing Proteins:  Antibodies

An important group of proteins that serve as reagents in cell biology is antibodies.  Immunoglobulin G (IgG, γ-globulin) is the most common type of antibody molecule used.  The individual antibody molecule consists of 4 polypeptide chains, two large subunits and 2 small subunits, held together by disulfide bonds.  (see http://www.accessexcellence.org/RC/VL/GG/ecb/antibody_molecule.html for a diagram)  IgG is often drawn as a Y-shaped structure.  The two outstretched arms of the Y are the variable regions of the molecule.  The variable regions are specific for a certain antigen conformation.  The remainder of the molecule is constant from IgG to IgG.

To raise antibodies, a purified protein (the antigen) is injected into an animal, like a rabbit.  The rabbit’s immune system is challenged.  The B lymphocytes begin manufacturing IgG molecules.  A particularly lymphocyte makes IgG with a particular variable region that will recognize a certain site on the antigen.  A second lymphocyte also makes IgG, but these IgG molecules have a different variable region that recognizes a different site on the antigen.  Overall, a mixture of IgG molecules are produced that recognize several sites on the antigen.  This mixture of IgG molecules is referred to as polyclonal antibodies.

Monoclonal antibodies are produced by a hybridoma.  An animal, usually a mouse, is immunized with an antigen.  In this case, the antigen does not have to be pure.  Once the mouse shows an immune response, its spleen is removed.  Lymphocytes are isolated from the spleen.  The lymphocytes cannot grow in culture, but if each is fused to a myeloma cell (which is cancerous), the hybrid cell (hybridoma) can grow in culture.  In culture, the hybridoma manufactures just one type of antibody molecule.  The individual hybridoma cultures are checked for specificity of the monoclonal antibody produced.  Therefore, any cultures that make antibodies to contaminants in the starting antigen can be ignored.  Only cultures making antibodies of interest would be maintained. 

Using Monoclonal vs. Polyclonal Antibodies
Monoclonal antibodies are specific for a particular antigen site.  This specificity cuts down on false positives.  There is a problem if the monoclonal is being used to screen for the whole antigen in organisms other than the one that provided the original antigen.  The particular site recognized by a monoclonal could be missing.  Alternatively, the site might not be recognizable following protein denaturation.  So, a particular monoclonal antibody could be useful for identifying the antigen with ELISA but not with a western blot (see below).  Polyclonal antibodies, with their mixture of recognition sites, increase the odds of recognizing at least one site on an antigen, no matter the source or the denaturation state.  The problem here is that even if IgG is purified from serum, that mix of IgG molecules contains all of the IgG the immunized animal made, including IgG molecules that recognize other antigens.  So, the chance of a false positive increases.  To control for that, duplicate analyses are done with pre-immune serum which is collected from the animal prior to immunization with the antigen.


For most applications, such as western blots and ELISA,  the antibody raised against a particular antigen is used as a primary antibody.  To see if that antibody has bound to something, the antibody might be tagged with an enzyme, a fluorescent dye, or a radioactive element.  The attachment of the tag requires a chemical reaction that might denature some of your primary antibody or alter the binding affinity to the antigen.  Therefore, the tags are usually attached to a secondary antibody.  The secondary antibody is raised in an animal different from the first, such as a goat.  The antigen in this case if IgG from the animal of the primary antibody.  The second animal raises antibodies to the first animal’s IgG that will mostly recognize the constant portion of the IgG molecule.  The secondary antibodies are usually raised in a larger animal, so there is a lot of secondary antibody available.  Pure antigen is also easily available as there is an affinity purification technique for IgG.  If some of the secondary antibody is denatured while attaching tags, it’s not as tragic as it would be if primary antibody were denatured.  (Also, solutions of untagged primary antibody can be re-used in some applications.)

Recognizing Proteins:  Lectins

Lectins are carbohydrate-binding proteins.  Many are bivalent or tetravalent, having two or four binding sites.  This allows a single lectin to bind to sugars on two or more different glycoproteins or glycoplipids.  If the glycosylated molecule is in the plasma membrane of a cell, lectins can link multiple cells together, causing them to clump or agglutinate.  Several blood typing procedures rely on the specific agglutinating ability of lectins to identify red blood cell antigens.  In fact, agglutination of red blood cells is often used to assess and screen for lectin activity.  Consequently, plant lectins are called phytohemagglutinins, particularly in the older literature.

Like enzyme active sites, the sugar-binding site of a lectin is specific for one sugar (or two very similar sugars).  Thus lectins are used extensively in cell biology to identify sugar residues on the exterior of cells, as absorbents for purification of glycoproteins, and as mitogens (molecules that stimulate mitosis).  

Lectins have been found in a variety of organisms, but the prime lectin search area is still plants.  Seeds in general are higher in lectins than other plant parts, with legume seeds being particularly rich in these proteins.  As much as 4% of the seed protein (by weight) can be lectin.  

Protein Separations:  Electrophoresis

Polyacrylamide gel electrophoresis (PAGE) is a convenient method for separating mixtures of proteins.  With sensitive staining procedures, ng quantities of protein can be examined.  PAGE can be performed under denaturing and nondenaturing (native) conditions.

Gels run under nondenaturing conditions are run either alkaline or acidic pH.  The separation takes advantage of the charges on the proteins from the R groups on the amino acids.  The advantage of running a native gel is that many enzymes can be assayed within the gel matrix, allowing for identification of particular proteins by their activity.

The most common denaturing agent is SDS (sodium dodecyl sulfate).  SDS is an anionic detergent that coats proteins and gives them a uniform charge to mass ratio.  The proteins are also uncoiled and acquire a rod shape.  Thus, proteins treated with SDS and then subjected to an electric field will migrate toward a positive pole on the basis of size, with the smallest proteins moving the fastest.  A reagent that disrupts disulfide bonds, such as 2-mercaptoethanol, is also added with the SDS so that the polypeptide chains uncoil completely and polypeptides joined by interchain disulfide bonds are released from each other.

Sometimes the powerful ability of SDS/PAGE to separate proteins in complex mixtures is coupled with a technique to identify a specific protein in the mixture.  This technique is Western blotting.  Following SDS/PAGE, the proteins are electrophoretically transferred to a special type of paper, usually a nitrocellulose (NC) membrane.  (This is the same material you used for the celery tissue prints.)  After the proteins are affixed to the membrane, the membrane is then probed with antibody against a particular protein.  This is the primary antibody.  To detect whether or not the primary antibody has bound to or "recognized" a particular protein on the NC membrane, a secondary antibody linked to an enzyme (usually horse radish peroxidase [HRP] or alkaline phosphatase [AP]) is used.  The enzyme is allowed to act on a substrate and release a colored, insoluble product.  This product will precipitate on the NC membrane and indicate where a primary antibody bound to a particular protein in the original mixture separated by SDS/PAGE.  This same protein detection technique can be done with lectins to identify glycoproteins by their sugar moieties.

pictures of stained and immunoprobed gels
outline from Plant Genetics on antibodies and blotting techniques  (pdf version)

Protein Separations:  Isoelectrofocusing (IEF) and 2-Dimensional (2-D) Electrophoresis

The primary way in which proteins are separated for analysis in the rapidly expanding field of proteomics is a method that has been around for a while: 2-D electrophoresis. In 2-D electrophoresis, proteins are first separated by charge (due to differences in the amino acid compositions of various proteins) and then by size.  The separation by charge uses isoelectrofocusing (IEF).  In IEF, a protein sample is placed on a gel that contains ampholytes.  The ampholytes form a pH gradient when a current is run through the gel.  Proteins will migrate through the gel until they “focus” at their  respective pI points.  (pI = the pH at which a protein is neutral) Once the IEF gel is done, the gel itself is incubated in a buffer that denatures proteins and then is placed on top of a second gel that contains SDS.  Electrophoresis then proceeds as it would for SDS/PAGE.

The individual protein spots can be cut out of the gel and microsequenced.  The sequences can be compared to those in data bases to identify the proteins and their functions.  The proteins in a  2-D gel can also be transferred to nitrocellulose and probed with antibodies or lectins as described above.

ELISA (Enzyme-linked Immunosorbent Assay)

While we’re on the subject of using antibodies to identify proteins, we’ll look at a technique that uses antibodies to quantify a particular protein in a mixture:  ELISA (Enzyme-linked Immunosorbent Assay).  There are a number of procedures for doing ELISA,  I’ll just go over a direct ELISA, which isn’t much different from probing a western blot.  ELISAs are usually done in 96-well (8x12) microtiter plates.  Proteins stick to the plastic that the plates are made of.  First, coat some of the wells with different dilutions of a solution that you want to test for the protein of interest (antigen).  Coat different wells with known concentrations of the antigen to serve as a standard curve.  First probe the wells with a primary antibody and then with an enzyme-linked secondary antibody.  The enzyme on the secondary antibody is allowed to act on a substrate that will release a soluble, colored product.  The product can be quantified with a microplate reader, which is essentially a spectrophotometer.  The amount of product is proportional to the amount of secondary antibody that bound, which is proportional to the amount of primary antibody that bound, which is proportional to the amount of antigen present in the sample.

Nucleic Acids
DNA and RNA:  negatively charged; will migrate toward the positive pole in an electric field
uniform mass:charge distribution; therefore, migration will be by size, with the smaller molecules migrating further and faster than the larger molecules.
Gel matrix:  agarose or polyacrylamide.  
Polyacrylamide –  generally reserved for very small nucleic acid molecules or sequencing gels.  Agarose – used to separate large fragments, products of DNA restriction digests.  Specific DNA or RNA molecules can be removed from an agarose gel for subsequent amplification (PCR), cloning, in vitro translation, etc.

Nucleic acids can be transferred from agarose gels to nitrocellulose, nylon, or PVDF and probed with oligonucleotides,  DNA, or RNA.  DNA was the first molecule to be transferred from a gel to a solid support.  The technique was created by E. M. Southern and became known as Southern blotting.  When RNA molecules were transferred, the technique was named for the opposite compass point, northern blotting.  Protein transfers were left to be western blots.

Next topic ==>  Protein Separations:  Chromatography

Links

http://faculty.plattsburgh.edu/donald.slish/Electrophoresis.html

Background on immunology:

http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookIMMUN.html

http://textbookofbacteriology.net/immune.html

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1 October  2002.  Links checked 15 May 2009.