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Dr. Bob is Robert Hsiung, MD,
bob@dr-bob.orgShown: posts 1 to 9 of 9. This is the beginning of the thread.
Posted by iforgotmypassword on February 10, 2008, at 13:07:40
has this been investigated thoroughly in any way? it practically sounds like a whole other branch of science i don't know about.
could theoretically someone's problems be due to antibodies at receptors and we haven't established their affinity, and just can't study every imaginable antibody this way. can it be simpler than just autoimmune effects on whole cells (as proposed in MS i think)?
i am wondering if this is the real issue in lyme disease. it's ability to be treatable with long-term antibiotics seems like it may be limited. i don't know anyone with the diagnosis who has gotten well.
Posted by Phillipa on February 10, 2008, at 13:07:40
In reply to affinity of long proteins, antibodies at receptors, posted by iforgotmypassword on February 10, 2008, at 12:01:45
I have chronic lymes but the docs I've seen say I always will test postive for Western blot. But the say the disease is not affecting me. I know that ANA is elevated in both lymes and autoimmune diseases as my hasimotos thyroiditis is one but my Ana is down and when lymes was active was 2850 or some outrageous high number like that hence they did MRI's and spinal taps to rule out MS and lupus and the others. Love Phillipa
Posted by Racer on February 10, 2008, at 13:07:40
In reply to Re: affinity of long proteins, antibodies at receptors » iforgotmypassword, posted by Phillipa on February 10, 2008, at 12:42:55
> the docs I've seen say I always will test postive for Western blot.
The Western blot is a type of test, so you'd test positive FOR Lyme disease ON a Western blot.
Posted by Phillipa on February 10, 2008, at 13:15:29
In reply to Re: affinity of long proteins, antibodies at recep » Phillipa, posted by Racer on February 10, 2008, at 13:07:40
Yes I know the docs had me on Iv antibiotics when first appeared and then oral for three years. The one up here said I'd had enough antibiotics when the health dept called and said my Western blot was positive. Called the rheumatologist here he said refer the health dept to him as I would always test positive. Was also told that by the infection control specialist who treated me in the hospital. Phillipa
Posted by Phillipa on February 10, 2008, at 13:25:52
In reply to affinity of long proteins, antibodies at receptors, posted by iforgotmypassword on February 10, 2008, at 13:07:40
This is just wiki but maybe the experts can figure it out. Love Phillipa hope this is what you are looking for as now I may have mad cow disease just joking.
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A Western blot.A western blot (alternately, immunoblot) is a method to detect a specific protein in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) (Figure 1) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein. There are now many reagent companies that specialise in providing antibodies (both monoclonal and polyclonal antibodies) against many thousands of different proteins. Commercial antibodies can be expensive, though the unbound antibody can be reused between experiments. This method is used in the fields of molecular biology, biochemistry, immunogenetics and other molecular biology disciplines.Other related techniques include using antibodies to detect proteins in tissues and cells by immunostaining and enzyme-linked immunosorbent assay (ELISA).
The method originated from the laboratory of George Stark at Stanford. The name western blot was given to the technique by W. Neal Burnette[1] and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern. Detection of RNA is termed northern blotting.
Contents [hide]
1 Steps in a western blot
1.1 Tissue preparation
1.2 Gel electrophoresis
1.3 Transfer
1.4 Blotting
1.5 Detection
1.5.1 Two step
1.5.2 One step
1.6 Analysis
1.6.1 Colorimetric detection
1.6.2 Chemiluminescence
1.6.3 Radioactive detection
1.6.4 Fluorescent detection
1.7 Secondary probing
2 2-D Gel Electrophoresis
3 Medical diagnostic applications
4 Protocols
5 References
6 See also
6.1 Related links
[edit] Steps in a western blot[edit] Tissue preparation
Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. Cells may also be broken open by one of the above mechanical methods. However, it should be noted that bacteria, virus or environmental samples can be the source of protein and thus western blotting is not restricted to cellular studies only.Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes.
A combination of biochemical and mechanical techniques including various types of filtration and centrifugation can be used to separate different cell compartments and organelles.
[edit] Gel electrophoresis
Main article: Gel electrophoresis
The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel.By far the most common type of gel electrophoresis employs polyacrylamide gels and buffers loaded with sodium dodecyl sulfate (SDS). SDS-PAGE (SDS polyacrylamide gel electrophoresis) maintains polypeptides in a denatured state once they have been treated with strong reducing agents to remove secondary and tertiary structure (e.g. S-S disulfide bonds to SH and SH) and thus allows separation of proteins by their molecular weight. Sampled proteins become covered in the negatively charged SDS and move to the positively charged electrode through the acrylamide mesh of the gel. Smaller proteins migrate faster through this mesh and the proteins are thus separated according to size (usually measured in kilo Daltons, kD). The concentration of acrylamide determines the resolution of the gel - the greater the acrylamide concentration the better the resolution of higher molecular weight proteins. Proteins travel only in one dimension along the gel for most blots.
Samples are loaded into wells in the gel. One lane is usually reserved for a marker or ladder, a commercially available mixture of proteins having defined molecular weights, typically stained so as to form visible, coloured bands. An example of a ladder is the GE Full Range Molecular weight ladder (Figure 1). When voltage is applied along the gel, proteins migrate into it at different speeds. These different rates of advancement (different electrophoretic mobilities) separate into bands within each lane.
It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.
[edit] Transfer
In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or PVDF. The membrane is placed on top of the gel, and a stack of tissue papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this "blotting" process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their non-specific protein binding properties (i.e. binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probings.The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie or Ponceau S dyes. Coomassie is the more sensitive of the two, although Ponceau S's water solubility makes it easier to subsequently destain and probe the membrane as described below.
[edit] Blotting
Since the membrane has been chosen for its ability to bind protein, and both antibodies and the target are proteins, steps must be taken to prevent interactions between the membrane and the antibody used for detection of the target protein. Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein - typically Bovine serum albumin (BSA) or non-fat dry milk (both are inexpensive), with a minute percentage of detergent such as Tween 20. The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached. Thus, when the antibody is added, there is no room on the membrane for it to attach other than on the binding sites of the specific target protein. This reduces "noise" in the final product of the Western blot, leading to clearer results, and eliminates false positives.
[edit] Detection
During the detection process the membrane is "probed" for the protein of interest with a modified antibody which is linked to a reporter enzyme, which when exposed to an appropriate substrate drives a colorimetric reaction and produces a colour. For a variety of reasons, this traditionally takes place in a two-step process, although there are now one-step detection methods available for certain applications.
[edit] Two step
Primary antibody
Antibodies are generated when a host species or immune cell culture is exposed to the protein of interest (or a part thereof). Normally, this is part of the immune response, whereas here they are harvested and used as sensitive and specific detection tools that bind the protein directly.After blocking, a dilute solution of primary antibody (generally between 0.5 and 5 micrograms/ml) is incubated with the membrane under gentle agitation. Typically, the solution is comprised of buffered saline solution with a small percentage of detergent, and sometimes with powdered milk or BSA. The antibody solution and the membrane can be sealed and incubated together for anywhere from 30 minutes to overnight. It can also be incubated at different temperatures, with warmer temperatures being associated with more binding, both specific (to the target protein, the "signal") and non-specific ("noise").
Secondary antibody
After rinsing the membrane to remove unbound primary antibody, the membrane is exposed to another antibody, directed at a species-specific portion of the primary antibody. This is known as a secondary antibody, and due to its targeting properties, tends to be referred to as "anti-mouse," "anti-goat," etc. Antibodies come from animal sources (or animal sourced hybridoma cultures); an anti-mouse secondary will bind to just about any mouse-sourced primary antibody. This allows some cost savings by allowing an entire lab to share a single source of mass-produced antibody, and provides far more consistent results. The secondary antibody is usually linked to biotin or to a reporter enzyme such as alkaline phosphatase or horseradish peroxidase. This means that several secondary antibodies will bind to one primary antibody and enhances the signal.Most commonly, a horseradish peroxidase-linked secondary is used in conjunction with a chemiluminescent agent, and the reaction product produces luminescence in proportion to the amount of protein. A sensitive sheet of photographic film is placed against the membrane, and exposure to the light from the reaction creates an image of the antibodies bound to the blot.
As with the ELISPOT and ELISA procedures, the enzyme can be provided with a substrate molecule that will be converted by the enzyme to a colored reaction product that will be visible on the membrane (see the figure below with blue bands).
A third alternative is to use a radioactive label rather than an enzyme coupled to the secondary antibody, such as labeling an antibody-binding protein like Staphylococcus Protein A with a radioactive isotope of iodine. Since other methods are safer, quicker and cheaper this method is now rarely used.
[edit] One step
Historically, the probing process was performed in two steps because of the relative ease of producing primary and secondary antibodies in separate processes. This gives researchers and corporations huge advantages in terms of flexibility, and adds an amplification step to the detection process. Given the advent of high-throughput protein analysis and lower limits of detection, however, there has been interest in developing one-step probing systems that would allow the process to occur faster and with less consumables. This requires a probe antibody which both recognizes the protein of interest and contains a detectable label, probes which are often available for known protein tags. The primary probe is incubated with the membrane in a manner similar to that for the primary antibody in a two-step process, and then is ready for direct detection after a series of wash steps.
western blot using radioactive detection system
[edit] Analysis
After the unbound probes are washed away, the western blot is ready for detection of the probes that are labeled and bound to the protein of interest. In practical terms, not all westerns reveal protein only at one band in a membrane. Size approximations are taken by comparing the stained bands to that of the marker or ladder loaded during electrophoresis. The process is repeated for a structural protein, such as actin or tubulin, that should not change between samples. The amount of target protein is indexed to the structural protein to control between groups. This practice ensures correction for the amount of total protein on the membrane in case of errors or incomplete transfers.
[edit] Colorimetric detection
The colorimetric detection method depends on incubation of the western blot with a substrate that reacts with the reporter enzyme (such as peroxidase) that is bound to the secondary antibody. This converts the soluble dye into an insoluble form of a different color that precipitates next to the enzyme and thereby stains the nitrocellulose membrane. Development of the blot is then stopped by washing away the soluble dye. Protein levels are evaluated through densitometry (how intense the stain is) or spectrophotometry.
[edit] Chemiluminescence
Chemiluminescent detection methods depend on incubation of the western blot with a substrate that will luminesce when exposed to the reporter on the secondary antibody. The light is then detected by photographic film, and more recently by CCD cameras which captures a digital image of the western blot. The image is analysed by densitometry, which evaluates the relative amount of protein staining and quantifies the results in terms of optical density. Newer software allows further data analysis such as molecular weight analysis if appropriate standards are used. So-called "enhanced chemiluminescent" (ECL) detection is considered to be among the most sensitive detection methods for blotting analysis.
[edit] Radioactive detection
Radioactive labels do not require enzyme substrates, but rather allow the placement of medical X-ray film directly against the western blot which develops as it is exposed to the label and creates dark regions which correspond to the protein bands of interest (see image to the right). The importance of radioactive detections methods is declining[citation needed], because it is very expensive, health and safety risks are high and ECL provides a useful alternative.
[edit] Fluorescent detection
The fluorescently labeled probe is excited by light and the emission of the excitation is then detected by a photosensor such as CCD camera equipped with appropriate emission filters which captures a digital image of the western blot and allows further data analysis such as molecular weight analysis and a quantitative western blot analysis. Fluorescence is considered to be among the most sensitive detection methods for blotting analysis.
[edit] Secondary probing
One major difference between nitrocellulose and PVDF membranes relates to the ability of each to support "stripping" antibodies off and reusing the membrane for subsequent antibody probes. While there are well-established protocols available for stripping nitrocellulose membranes, the sturdier PVDF allows for easier stripping, and for more reuse before background noise limits experiments. Another difference is that, unlike nitrocellulose, PVDF must be soaked in 95% ethanol, isopropanol or methanol before use. PVDF membranes also tend to be thicker and more resistant to damage during use.
[edit] 2-D Gel Electrophoresis
2-dimensional SDS-PAGE uses the principles and techniques outlined above. 2-D SDS-PAGE, as the name suggests, involves the migration of polypeptides in 2 dimensions. For example, in the first dimension polypeptides are separated according to isoelectric point, while in the second dimension polypeptides are separated according to their molecular weight. The isoelectric point of a given protein is determined by the relative number of positively (e.g. lysine and arginine) and negatively (e.g. glutamate and aspartate) amino acids, with negatively charged amino acids contributing to a high isoelectric point and positively charged amino acids contributing to a low isoelectric point. Samples could also be separated first under nonreducing conditions using SDS-PAGE and under reducing conditions in the second dimension, which breaks apart disulfide bonds that hold subunits together. SDS-PAGE might also be coupled with urea-PAGE for a 2-dimensional gel.In principle, this method allows for the separation of all cellular proteins on a single large gel. A major advantage of this method is that it often distinguishes between different isoforms of a particular protein - e.g. a protein that has been phosphorylated (by addition of a negatively charged group). Proteins that have been separated can be cut out of the gel and then analysed by mass spectrometry, which identifies the protein.
Please refer to reference articles for examples of the application of 2-D SDS PAGE.
[edit] Medical diagnostic applications
The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample. Proteins from known HIV-infected cells are separated and blotted on a membrane as above. Then, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary anti-human antibody linked to an enzyme signal is added. The stained bands then indicate the proteins to which the patient's serum contains antibody.
A Western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE, commonly referred to as 'mad cow disease').
Some forms of Lyme disease testing employ Western blotting.
Posted by Racer on February 10, 2008, at 14:58:04
In reply to Re: affinity of long proteins, antibodies at recep » Racer, posted by Phillipa on February 10, 2008, at 13:15:29
Posted by Phillipa on February 10, 2008, at 20:04:15
In reply to I wasn't asking for information, but offering it (nm) » Phillipa, posted by Racer on February 10, 2008, at 14:58:04
Clicked your name by mistake apolgies this info was for others that might choose to read the thread. Info is knowledge. Phillipa
Posted by ShawnThomas on February 11, 2008, at 10:28:53
In reply to affinity of long proteins, antibodies at receptors, posted by iforgotmypassword on February 10, 2008, at 13:07:40
I found a report of an antigen on Borrelia burgdorferi causing myasthenia gravis by blocking the action of acetylcholine at nicotinic acetylcholine receptors (http://pubmed.gov/17366045). I do not believe that that this is common result of Borrelia burgdorferi infection.
A greater concern is a surface protein on Borrelia burgdorferi that can activate Toll-Like Receptor 2 (TLR2). See
http://www.jimmunol.org/cgi/content/full/163/5/2382According to Hirschfeld et al. (1999), "Lyme disease is caused by infection with the tick-borne spirochete Borrelia burgdorferi (1). A subacute inflammatory arthritis develops in 60% of individuals not treated at the time of the tick bite (2), and is associated with invasion of the joint tissue by spirochetes (3, 4). B. burgdorferi possess surface-associated proteins with the tripalmitoyl-S-glyceryl-cysteine (Pam3Cys)3 modification common to many bacterial species (5), and numerous inflammatory activities have been ascribed to the Pam3Cys modification (6, 7, 8, 9).
...
Our results demonstrate that TLR2 is required for bacterial lipoprotein signaling in two different TLR2-negative cell lines, U373 and 293, and that signaling requires the lipid modification."Also, you can search http://www.pubmed.gov for "Toll-Like Receptor 2"[Mesh] AND brain
This receptor is definitely involved in inflammation in the brain and elsewhere.
Shawn
Posted by Phillipa on February 11, 2008, at 12:07:47
In reply to Re: affinity of long proteins, antibodies at recep » iforgotmypassword, posted by ShawnThomas on February 11, 2008, at 10:28:53
Shawn I guess I'm very lucky that my untreated don't know for how long but probably years didn't leave me with the lyme's arthritis. For how many years will I test postivie with Western blot for lymes? And as a side note a neighbor in early 70's has been unsuccessfully treated for gout of all lower extremities and can't walk at all been on steroids not good and pain killers. Did a search last night as his wife said it had gout and ended up with a lyme arthritis and his creatinine is also becoming elevated not good. I replied to her that a sed rate when taking him in to specialist again might be a good place to start. Do you agree? And thankfully my spinal taps were negative both times but the spirochettes go into hiding in body tissues. Heart ect so hope mine are gone and hopefully all others are gone too. Thanks for the info Phillipa
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