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 Western Blotting Report

Analytical biochemistry has a now well-established and very important procedure occupied by Western blots (Immuno-blots or protein blots). This successful niche in the lab has been established primarily because of their ease of use, accuracy, and visually orientated nature. Western blotting uses electrophoresis and immunology together to help detect the presence of specifically desired proteins. The technique is also helpful in ascertaining quantity of the target protein, the molecular size or weight of the protein, and its purity. Western blots can be visually orientated by using antibodies coupled with chemiluminescent flags, enzymes or radioactive reagents. The Chemiluminescent and radioactive reagent sites can be developed by Autoradiography to visualise the presence and locality of the target protein. The enzyme-linked reagents can be photographed or seen in real time by the colours that they produce when reacted with the proper reagents.

The formation of hybrids in solution is of little experimental value - if you mix a solution of DNA with a solution of radioactive probe, you end up with just a radioactive solution. You cannot tell the hybrids from the non-hybridised molecules. For this reason, you must first physically separate the mixture of molecules to be probed on the basis of some convenient parameter. These molecules must then be immobilised on a solid support, so that they will remain in position during probing and washing. The probe is then added. The non-specifically bound probe is removed, and the probe is detected. The place where the probe is detected corresponds to the location of the immobilised target molecule.

Practicability, accuracy, and the easily observed results of the Western Blot have made its use indispensable in almost all the biologically orientated fields. Western blotting is used in the Animal Sciences and in the Food Sciences to demonstrate the presence and quantity of target proteins in the animals and plants that are studied. In Zoology and Botany it is used much the same. Western blotting is used in Immunology and Microbiology to show which antibodies bind to which proteins in the studied organism. In Biochemistry and Molecular Biology, it is used to help develop and discover new proteins. A Western blotting makes it possible to find a protein in a complex mixture, which would otherwise be a proverbial needle in a haystack. This technique is used advantageously in the cloning of genes (Stryer, 1996). In each of the above mentioned fields it is used in many other different ways.

Prior to the invention of Western blots, SDS-PAGE (Sodium Dodecylsulfate-polyacrylamide gel electrophoresis) and immuno-chemical detection were used. Various derivations of SDS-PAGE have been developed, in both one and two dimensions. These processes separate proteins apart by mass and/or isoelectric focusing. Immuno-chemical detection uses the understanding of antigen-antibody binding, similar to immuno-precipitation, to target the proteins in Western Blots. At first these two technologies were combined to stain polyacrylamide gels with antibodies (Burridge 1976;Showe et al. 1976). This technique however is tricky because diffusion occurs best when polyacrylamide concentrations are very low. This weakens the gel and allows greater diffusion of targeted protein out of the gel thus reducing resolution. Over the last two decades a method has evolved in order to analyse specific target proteins by using specific antibodies designed to target the protein of research. This includes the discovery that proteins could be transferred to a membrane, nitro-cellulose usually is most preferential, but also chemically activated paper (Diazobenzyloxymethyl (DBM) paper) and nylon, or Zeta-bind nylon, (Gershoni and Palade 1983; Towbin and Gordon 1984; Beisiegel 1986). The transfer of the protein to the membrane can be mediated by passive diffusion (48 hours). Electrophoretic transfer, either semi-dry or wet speeds up the process remarkably (As little as 4 hours) (Towbin et al 1979, Burnette 1981). A process called vacuum blotting was also introduced which is also a very inexpensive, straightforward, reproducible and extremely fast (Peferoen et al 1982).

Source: Adapted from (http://esg-www.mit.edu:8001/esgbio/rdna/rdna.html), Southerns, Northerns, Westerns, & Cloning: "Molecular Searching" Techniques; Written by Brian White, MIT.

Western blot protocols can be divided into six steps (Kaspar, 1997).

1. Antigen preparation
2. Electrophoresis
3. Transfer of proteins to a membrane
4. Blocking of non-specific binding sites on the membrane
5. Antibody addition
6. Enzymatic or radioactive detection

Antigen preparation - The cells of the organism and indeed tissue (In the case of multicellular organisms) of interest must be lysed in order to release the multitude of proteins contained inside the cytosols and membranes of the cells. Before lysing the cells, it is necessary to add a solution containing protease inhibitors, such as PBS, so that upon lysis the proteases don’t destroy the proteins. Lysis can be induced by freeze/thaw cycles of 5 minutes in dry ice and ethanol and 5 minutes in a 37 ° C water bath. The membrane and polynucleotide components may then be removed.

Electrophoresis - An appropriate SDS/PAGE loading dye (Includes dye markers) is added to the protein solution. The proteins can then be separated by electrophoresis by either one or two-dimensional electrophoresis on a 12.5% SDS-PAGE gel. Pre-stained standards can be used to mark the progression and the efficiency of the electrophoresis. It is usually more convenient to use a discontinuous SDS-PAGE gel, using two different polyacrylamide concentrations to create stacking and sequencing sections of the gel.

Transfer of proteins to a membrane - The gel, nitro-cellulose paper and Whatman paper are washed with a transfer buffer containing glycine, Tris, SDS, and methanol. A western transfer apparatus is prepared by sandwiching two pieces of Whatman paper, the nitro-cellulose membrane, the SDS-PAGE gel, and two more pieces of Whatman paper, in that order, between two wet porous pads. This sandwich formation is pressed between the two plastic panes of the western transfer apparatus with the nitro-cellulose nearest to the positive electrode of the apparatus. The apparatus is run overnight with about 30 volts electricity and stirring in a cold room.

Blocking of non-specific binding sites on the membrane - The non-specific binding sites are filled by incubating the membrane overnight in a buffer-salt solution, such as TBST, containing dry non-fat milk such as Marvel^TM (about 5%). An alternative blocking solution is TWEEN 20 (Batteiger, 1982).

Antibody addition - Antibodies are prepared by injecting the target protein into an animal (e.g. goat, rabbit or mouse) and removing the serum produced by the introduction of the protein antigen. When two different antibodies are produced, they must come from different animals. The membrane must again be washed twice with a buffer-salt solution, such as TBST again, to prepare the membrane for antibody addition. The membrane is then put into a heavy plastic bag or plastic tube and treated with the first antibody solution (containing PBS). This first antibody binds to the specified protein. Again, the membrane is then washed as before. The second antibody is added to the membrane as before. This antibody binds to the first antibody. This second antibody can also be bound to an enzyme that changes the colour of a substrate or have a radioactive label. The membrane is again washed in a solution such as TBST and also in Western Developing Buffer (contains NaCl, MgCl₂, and Tris).

Enzymatic or radioactive detection – A Detection with enzyme-labelled antibodies is the most popular method, normally using horseradish peroxidase or alkaline phosphatase-couple antibodies, and a range of soluble substrates that yield insoluble coloured proteins (Harlow, 1988). The results are easy to obtain, immediate, visible and do not require special equipment. To be able to quantitate the results, it is better to use radioactive labels. This can be done using timed exposure to Autoradiography film or by excising bands out of the membrane and performing a radioactive count. An alternative approach would be to bind the second antibody with fluorescein. When this molecule absorbs UV light, it fluoresces in a green colour (Towbin, 1979)

Source: Ref. #16

1. Agarose plug:
   1-% agarose dissolved in 1x Resolving gel buffer.
   (Make 50 ml and melt it, as required. Re-add water to maintain agarose concentration)
2. Resolving gel: 24 ml of a 9% gel
   5.4 ml 40% acrylamide/bis-acrylamide (29:1 mix)
   3 ml 8x Resolving gel buffer
   15.6 ml water
   12 µl TEMED
   60 µl 20% ammonium persulphate
3. Stacking gel: 8 ml
   1 ml 40% acrylamide/bis-acrylamide (29:1 mix)
   2 ml 4x Stacking gel buffer
   5 ml water
   8 µl TEMED
   21.6 µl 20% ammonium persulphate

1. Assemble the glass plates and spacers (1.5 mm thick).
2. Pour an agarose plug (1-2 mm).
3. Pour the running gel to about 1 cm below the wells of the comb (~20 ml).
4. Seal with 1 ml water-saturated 1-butanol.
   (Can stop here and leave gel as is overnight if you want.)
5. When gel has set, pour off the butanol and rinse with deionised water.
6. Pour the stacking gel (~5 ml) and insert the comb immediately.
7. When the stacking gel has set, place in gel rig and immerse in buffer.
8. Prior to running the gel, flush the wells out thoroughly with running buffer.

1. After flash spinning the samples, load into the wells.
2. Be sure to use markers.
   We use 15 µl Bio-Rad Kaleidoscope Pre-stained Standards #161-0324 directly.
3. Run with constant current (35 - 37 mA with voltage set at > 300 V).
4. Usual running time is about 2.5 hr.

Using pre-cast gels (Ready Gels from Bio-Rad):

1. Assemble gel in gel rig.
2. Prepare protein samples (10 µg will suffice).
3. Use 5 µl of Kaleidoscope standard.
4. Run at 200 V (constant voltage) for 30 min.

1. Cut a piece of PVDF membrane (Millipore Immobion-P).
2. Wet for about 30 min in methanol on a rocker at room temp.
3. Remove methanol and add 1x Blotting buffer until ready to use.

Membrane transfer

1. Assemble "sandwich" for Transblot.
2. Pre-wet the sponges, filter papers (slightly bigger than gel) in 1x Blotting buffer.
   Sponge - filter paper - gel - membrane - filter paper - sponge
3. Transfer for 1 hr at 1 amp at 4°C on a stir plate.
   Bigger proteins might take longer to transfer.
   For the Mini-Transblot, it's 100 V for 1 hr with the cold pack and pre-chilled buffer.
4. When finished, immerse membrane in Blocking buffer and block overnight.

1. Incubate with primary antibody diluted in Blocking buffer for 60 min at room temp.
2. Wash 3 x 10 min with 0.05% Tween 20 in PBS.
3. Incubate with secondary antibody diluted in Blocking buffer for 45 min at room temp.
4. Wash 3 x 10 min with 0.05% Tween 20 in PBS.
5. Detect with Amersham ECL kit.

1. Rinse blot off with 0.05% Tween 20 in PBS.
2. Put blot into Kapak bag cut to slightly bigger size than blot.
3. Add about 5 to 10 ml Stripping buffer.
4. Remove as much air as possible and seal bag.
5. Immerse into 80°C water bath and incubate for 20 min.
6. Rinse blot off with 0.05% Tween 20 in PBS.
7. Block for about 1 hr with 5% BSA/Tween 20, or overnight with 3% BSA/Tween 20.

Buffers used for Western Blots

Lysis buffer:

0.15 M NaCl

5 mM EDTA, pH 8

1% Triton X100

10 mM Tris-Cl, pH 7.4

Just before using add: 1:1000 5 M DTT

1:1000 100 mM PMSF in iso-propanol

1:1000 5 M aminocaproic acid

2x sample buffer:

130 mM Tris-Cl, pH8.0

20% (v/v) Glycerol

4.6% (w/v) SDS

0.02% Bromophenol blue

2% DTT

8x Resolving gel buffer: 100 ml

0.8 g SDS (add last)

36.3 g Trizma base (= 3 M)

Adjust pH to 8.8 with concentrated HCl

4x Stacking gel buffer: 100 ml

0.4 g SDS (add last)

6.05 g Trizma base (= 0.5 M)

Adjust pH to 6.8

10x Running buffer: 1 L

30.3 g Trizma base (= 0.25 M)

144 g Glycine (= 1.92 M)

10 g SDS (= 1%)--add last

10x Blotting buffer: 1 L

30.3 g Trizma base (= 0.25 M)

144 g Glycine (= 1.92 M)

pH should be 8.3; do not adjust

1x Blotting buffer: 2 L

400 ml Methanol

200 ml 10x Blotting buffer

1400 ml water

Blocking buffer: 0.5 L

3% Bovine serum albumin (Fraction V)

Make up in PBS and sterile filter.

Then add 0.05% Tween 20.

Keep at 4°C to prevent bacterial contamination.

Stripping buffer: 0.5 L (sterile filter solution and keep at 4°C)

0.2 M Glycine, pH 2.5

0.05% Tween 20

The protein used was a MAPK (Mitogen Activated Protein Kinase) Isolated from rat brain. This was isolated from lysates previous to the workshop on an SDS Gel. The blot was performed on the same day and the nitro-cellulose paper was kept until the day we used it. The robust nature of the blotting makes it a reliable and near fail-safe technique for all levels of competence.

ECL was used to detect the immuno-reactive proteins. The prepared nitro-cellulose paper was placed in an autoradiograph cassette for sixty seconds once activated (See Materials and Methods). Below is a copy of the autoradiograph, annotated to give the band definitions observed.

The distances travelled by the separated bands of protein species were Below is the graph upon which the distances travelled by the various protein species are mapped:

We can see that the lineage is indeed proportinal and both species of the MAPK separation have given positive results………

Western blotting is currently being used in many extensive ways. Western blots are currently being used in cloning techniques. Scientists use proteins to prepare specific antibodies to place in an expressed cDNA library in order to target the gene or cDNA that they wish to clone. Western blotting is also being used to determine the sequence of specific genes of various organisms in several different genome projects (e.g. The Human Genome Project). Western blots are currently being used along with ELISA (enzyme-linked immunosorbent assay) to determine the presence of HIV in patients. This can be done with numerous types of diseases of viral, bacterial, genetic, and parasitic (e.g. dipetalonema, amoeba, mycoplasma, chlamydia, spirochetes, measles, herpes, hepatitis, poliovirus, multiple sclerosis); even the detection of proteins involving ageing, allergies, and fertility (Towbin 1984). In the same article Towbin concludes: New findings using the immuno-blot methodology are likely to be made in the future. The techniques developed may use the detection of pathological electrophoretic variants; the detection of presence or absence of pathological immuno-reactivity amongst electrophoretically separated antigens. These analyses may well find applications in routine diagnostics.

Below are some research articles eluded to in principle above but show in detail how the western blot analysis is widely used in clinical biochemistry. Thus in screening for diseases and analysis of as-yet unknown diseases. Hopefully these examples will demonstrate more conclusively how powerful a system western blotting is.

Lyme disease, known as LD or Lyme Borreliosis, is the most common tick-borne infection in the United States. Lyme disease is a multi-system illness that primarily affects the skin, heart, joints, and nervous system. Lyme disease is a public health concern because the distribution of the disease is expanding and is persistent symptoms, permanent tissue injury and disability often complicate manifestations of the disease.

In 1982, a spiral shaped bacterium called Borrelia burgdorferi was obtained from ticks in New York. This same organism was then isolated from blood specimens from people with Lyme Disease. This bacterium is transmitted to humans by the bite of an infected tick. Lyme disease occurs worldwide. Most cases are reported from temperate regions and coincide with the distribution of the principal vector ticks - Ixodes pacificus (Pacific NW US) and Ixodes dammini (East and Central US). There are more than 10,000 cases being reported annually to the Centre for Disease Control and Prevention. The cause of the illness is a bacterium called Borrelia burgdorferi.

The most common sites of the skin lesion are on the thigh and groin. Up to 80% of patients with EM, have associated systemic complaint, including fatigue, myalgias, arthralgias, headache, fever, and/or chills, stiff neck. Lesions promptly resolve with antibiotic therapy, although, if untreated, they may last months and even years.

Early detection of Lyme disease is possible and treatment through antibiotics can prevent complications of the disease. The diagnosis of Lyme disease relies on the presence of characteristic clinical features and supporting serological test results. Diagnosis of Lyme disease is through detection of the bacterium from tissue or body fluid or antibodies to the bacterium. Antibodies usually develop within 2 to 4 weeks after the infected tick bite. These antibodies peak 6 to 8 weeks after the initial infection and resume to normal levels after 4 to 6 months. A Lyme ELISA test also can be used to detect the immunological response. Although serological testing may yield negative results during the first several weeks of the infection, most patients have a positive antibody response to B. burgdorferi after that time. For serological analysis in Lyme disease, the CDC recommends a 2-step approach in which ELISA first test samples and equivocal or positive results are then tested by Western blotting. The most common problem in diagnosis is to distinguish late stage Lyme disease from chronic fatigue syndrome or fibromyalgia. This difficulty is compounded because a small percentage of patients develop these chronic pain or fatigue symptoms in association with or soon after Lyme disease. A physician uses the intensity of the symptoms and their location to distinguish between the 2 disease.

In a 1997 paper entitled "Simultaneous ELISA and Western Blot Testing In Evaluation of Patients For Suspected Lyme Disease" (See References), the writers presented findings on simultaneous Elisa and Western Blot testing in a practice specialising in the diagnosis and treatment of Lyme disease.

Elisa and Western Blot results on all 62 new patients to the practice, during the last quarter of 1996, were retrospectively reviewed. A positive Western Blot is defined by the "CDC" criteria as the presence of at least two specific IgM bands, or the presence of at least 5 specific IgG bands. A suspicious Western Blot is defined by this office as the presence of any "CDC" specific band that is present in either the IgM or the IgG band lines, or the presence of 31 (OSP A), or 34 (OSP B) kilodalton bands in either the IgM or the IgG band lines. A borderline Elisa is defined as reactivity within 2 to 3 standard deviations from the control. Blood work from the 62 new patients were sent to two independent Immuno-pathology laboratories

16% (10 patients) tested for Lyme disease resulted in a positive Elisa and a positive Western Blot. 10% (6 patients) tested for Lyme disease resulted in a positive Elisa and a suspicious Western Blot. 3% (2 patients) tested for Lyme disease resulted in a borderline Elisa and a positive Western Blot. 5% (3 patients) tested for Lyme disease resulted in a borderline Elisa and a suspicious Western Blot. 21% (13 patients) tested for Lyme disease resulted in a negative Elisa and a positive Western Blot. 45% (28 patients) tested for Lyme disease resulted in a negative Elisa and a suspicious Western Blot. 0% (0 patients) tested for Lyme disease resulted in a negative Elisa and a negative Western Blot.

In a population of patients with clinical suspicion of Lyme disease, Western Blotting showed a high yield of bands having relevance to Lyme disease. As high as 21% of patients with totally negative Elisas demonstrated fully diagnostic Western Blots which are quoted to satisfy the "stringent CDC criteria" intended to define a "Case" for epidemiological surveillance purposes. Many other patients showed suspicious bands not expected to be found in persons not having been exposed to B. burgdorferi. A single suspicious band (as defined above), and particularly one or more bands is frequently indicative of an expansion of significant bands over time and may serve as an early clue to the presence of Lyme Disease.

A two-tiered approach to testing, although standard for HIV, may be inappropriate in Lyme disease since Lyme Western Blotting is more specific than Elisa testing and my be more sensitive. Omission of Western Blotting in patients clinically suspected of having Lyme disease when Lyme Elisas are negative would have missed conclusive proof of Lyme disease in 21% of patients, and important clues to possible diagnosis in 63% or more. These findings suggest that patients whom are clinically suspected of having Lyme disease, simultaneous Lyme Elisa and Western Blotting should replace the presently recommended two-tier schema of testing.

Another paper from 1997 entitled "Amyloid ß-protein (Aß) Accumulation in the Leptomeninges during Ageing and in Alzheimer’s Disease" sought to demonstrate a link between Aß deposition and Alzheimer’s disease. Well-characterised two-site enzyme immunoassays showed that the crude leptomeninges (consisting of the pia matter, arachnoid matter, and leptomeningeal vessels [LV]) from aged control brains and brains affected by Alzheimer disease (AD) contain very high levels of amyloid ß-protein (Aß). To learn about the source of Aß, we carefully dissected out both leptomeninges (LM) and LV under a dissecting microscope and determined the levels of soluble Aß in each. The absence or presence of alpha-smooth muscle actin confirmed the purity of these dissected tissues representing LV by Western blotting. Surprisingly, the amounts of Aß in each dissected sample were nearly equivalent on a weight basis. In each compartment from aged controls the level of Aß1-42 was comparable to that of Aß1-40, while in AD brain Aß1-40 was a predominant species in both LM and LV. This use of the technique demonstrates how quantitative analyses can be performed.

They initially thought that such large amounts of Aß were derived exclusively from LV. However, a preliminary result unexpectedly suggested that leptomeninges (LM) themselves might contain significant levels of Aß comparable to those in LV on a weight basis. Tissue dissected specimens (~30 mg wet weight) were cut into small pieces with a sharp blade and homogenized in 19 volumes of Tris-saline (TS) containing protease inhibitor cocktail (see above) using a Teflon/glass motor-driven homogeniser for 20 strokes. The 5% homogenates were centrifuged at 100,000-x g for 15 min on a TL 100.3 rotor in a TLX centrifuge (Beckman, Palo Alto, CA). The resultant supernatants (~0.9 mg/ml) were diluted more than 5-fold with 20 rnM phosphate buffer (pH 7.0) containing 0.4 M NaCl, 2 rnM EDTA, 10% Block Ace (Dainippon, Tokyo, Japan), 0.2% bovine serum albumin, 0.075% CHAPS, and 0.05% NaN₃ (buffer EC), and subjected to the two-site EIA. The resultant pellets, after one wash, were further extracted with more than 100 volumes (to the initial tissue volume) of 70% formic acid. The homogenates were similarly centrifuged on a TL 100.3 rotor. The supernatant was neutralised with NaOH and trizma base, and applied to EIA.

A small portion of aorta (~25 mg wet weight) from a 47-year-old male was cut into small pieces with a sharp blade and homogenized in 19 volumes of TS containing protease inhibitor cocktail using a Teflon/glass motor-driven homogeniser for 20 strokes. The homogenates were centrifuged at 100,000 X g for 15 min on a TL 100.3 rotor. The supernatant was subjected to SDS-Polyacrylamide Gel Electrophoresis (PAGE) and Western blotting for alpha-smooth muscle actin. From 1 mg wet weight of aorta we obtained 17.8 µg of soluble proteins, and the proportion of ASMA was quantitated to be 6.15% of the total soluble protein (see below).

Aß BAN50 monoclonal antibodies were used in the present study and on Western blots of insoluble fractions from AD and aged brains, Aß43 is almost undetectable, as shown with BC65, a monoclonal antibody specific for Aß43 (unpublished data), and thus BC05-based EIA values can largely be considered to represent Aß42. Monoclonal antibodies to alpha-smooth muscle actin (ASMA; Sigma, St. Louis, MO), glial fibrillary acidic protein (GFAP; Sigma), polyclonal antibodies to von Willebrand factor (vWf; Dako, Glostrup, Denmark), and fibronectin (Sigma) were used for Western blotting.

For immuno-precipitation of Ads in soluble fractions of LM and LV, an antiserum raised against Aß1-40 (Bachem, Torrance, CA) was used. Using synthetic Aß1-40 and 1-42, we confirmed that the antiserum precipitated both species of A,B. After the LM supernatants were incubated with excess protein A agarose (Gibco, Gaithersburg, MD) at RT for 2 h and cleared by centrifugation, the antiserum was added to the supernatant at a dilution of 1:100 and the mixtures (600 µl) were incubated at 4°C overnight. Protein A agarose was added to the mixture, followed by incubation at RT for 4 h and brief centrifugation. The resulting pellets were rinsed 3 times with 1X RIPA buffer (50 mM Tris HCI, 0.15 M NaCI, 1% Nonidet P-40 and 0.1% SDS, pH 8). The final pellets were heated in Laemmli buffer and extracts were loaded onto a Tris-tricine gradient gel (MULTIGEL 15/25. Daiichi Pure Chemicals Co., Ltd. Tokyo, Japan). Separated proteins were subjected to Western blotting with BAN50 (see below).

In Western blotting for ASMA, GFAP, vWf or fibronectin, appropriate amounts of specimens were subjected to SDSPAGE and Western blotting. The protein concentrations in the samples, homogenates of cortices, LV and LM fractions, and cell lysates were determined with a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockfield, IL). Separated proteins were transferred onto nitro-cellulose membranes (0.22 Em pore size; Schleicher & Schnell, Germany) or polyvinylidene difluoride (PVDF) membrane (Nihon Millipore Ltd. Yonezawa, Japan) for 1 h in a semidry blot apparatus. The blots were immersed in 5% skim milk in TS (pH 7.6) containing 0.2% Tween 20 (TS-T) for 1 h at RT. After a rinse with TS-T, the blots were incubated with each monoclonal antibody to ASMA or GFAP, or each polyclonal antibody to vWf or fibronectin, for 1 h at RT at appropriate dilutions recommended by the manufacturer. After a second rinse, the blots were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG or anti-rabbit IgG for 1 h at RT. After a final wash, immuno-reactive proteins were visualised with an enhanced chemiluminescence (ECL) system (Amersham, Buckinghamshire, UK).

To assess the sensitivity of the ECL-coupled ASMA monoclonal antibody, the following experiment was done. Aliquots from the supernatant of aorta homogenate were subjected to SDS-PAGE and one portion of the gel was stained with Coomassie Brilliant Blue. Staining the gel allows on to see the level of remainder generic protein left in the gel. The relative amount of actin at 43 kDa in the supernatant was quantitated using a densitometer (Model GS-700 imaging densitometer; BioRad, Tokyo, Japan). The relative amount of actin in the homogenate supernatant was estimated to be 6.15 + 0.52%. Those separated proteins in the other portion of the gel were transferred to PVDF membrane, followed by Western blotting for ASMA. On the Western blot for ASMA, the immuno-reactive band at 43 kDa was detected in the sample containing 0.025 fig of protein from the supernatant, indicating that our system can detect 1.5 ng of ASMA (data not shown). This is the minimal estimate for the sensitivity because the vascular smooth muscle is known to contain other actin isoforms that do not react with the monoclonal antibody (in the case of the aorta the proportion of ASMA amounts to 60% of the total actin).

Based on the results of many immunocytochemical studies, it is generally thought that in crude leptomeninges, Aß accumulates exclusively in LV, resulting in CAA. The presence of bundles of amyloid fibrils in the media (smooth muscle layer) of the leptomeningeal vessel has been repeatedly confirmed. It was suggested that amyloid fibrils are synthesised in smooth muscles of the LV. In this way, the LV has long been known as the major site of Aß deposition: however, to our knowledge, no findings of Aß accumulation in leptomeninges have been reported, except in one case report on spinal amyloid angiopathy. This is presumably because the presence of Aß deposits in the leptomeningeal layer was rarely shown unequivocally; it is rather difficult to distinguish the true staining from artefacts that readily occur at the edges of tissue sections, and leptomeninges are usually only poorly preserved in conventional tissue processing. The great thing attribute of western blotting is that specific proteins can be probed and re-probed. Thus although the test is only as specific as the antibody, by using reprobing techniques with different antibodies specific to the same protein, chances of probing random or mis-targetted proteins becomes increasingly less significant.

In the present study, crude leptomeninges were taken from various areas; that Aß species predominance depends upon the brain region cannot be completely disregarded. Another unlikely explanation is that substantial amounts of Aß1-42 were washed out or altered during dissection in such a manner that BAN50 was no longer able to capture or BC05 was no longer able to detect. This does not alter the accepted accuracy of the western blot. Small proteins can be searched for with small diameter pore filtration paper, and the reverse for large proteins. The only phenomenon known to not produce results is when proteins "Blow through" because small proteins in combination with high currents can pass through large pores.

This was the first report that provides clear evidence that leptomeninges are a large reservoir of Aßs. The present findings, together with previous ones, strongly suggest that intracranial tissues can accumulate Aß during ageing; Aß accumulation may be an important characteristic of such tissues. It seems reasonable to postulate that preferentially deposited molecular species (Aß 40 or 42) may be defined by the cells involved in ß-amyloidogenesis. Many of the techniques are only supported because of the fact that western blots are incredibly reliable and support roles that no other technique can perform, i.e. searching for elusive proteins and giving relative quantitation.

A parisitology research paper " Analysis of Protein Profiles as a Tool to Differentiate Trypanosoma cruzi Isolates Obtained from Different Geographic Areas" was heavily dependant upon the research using western blotting techniques. T. Cruzi populations are heterogeneous and this can be a modulating factor for the clinical variability detected in isolates recovered from different geographical regions. This characteristic frequently makes difficult the interpretation of serological tests. Therefore, determination of major antigens among different isolates can contribute to the development of more accurate approaches.

Proteins from T. cruzi isolates obtained from patients living in two different Chagas disease endemic areas, located in Paraíba and Piauí states (Brazil) were analysed by SDS-PAGE and Immuno-blot. Protein extracts were prepared from epimastigotes forms harvested after 5 days of growth in LIT medium. Parasites were disrupted by freeze and thaw followed by sonication. The suspensions were centrifuged and the supernatant used as antigens. The preparations were treated with SDS and separated in 10% acrylamide gels by electrophoresis. Proteins in the gels were silver-stained and the protein profiles were analysed either by unaided visual inspection or by densitometric readings. Calculations of the similarities were done with or without the aid of specific computer programs. Protein profiles from 9-T. cruzi isolates obtained from Paraíba showed 14 to 27 bands with Mrs ranging from 14 to 105 kDa. In 8 of these isolates the protein profiles showed high similarity (unaided visual inspection), with minor quantitative and qualitative differences. The average of similarity coefficient for such isolates was about 70%. Two of them (PB 425 and PB 475) showed 100% similarity. When the Paraíba isolates were compared with the Y strain and VL 983 isolate, similarity was 50% or lower. One sample (PB 913) showed a more complex protein profile, which suggested a mixed parasite population. Further characterisation of this isolate is being done by limited dilution cloning and biological behaviour in Swiss mice.

Similar protein profiles were also observed in 10-T. cruzi isolates obtained from patients living in Piauí. The protein profiles were composed of 12 to 18 bands ranging from 18 to 110 kDa. The average of the similarity coefficient was around 85%. The Piauí isolates showed less than 60% of similarity when compared with the Y strain.

For immuno-blot analysis, T. cruzi antigens from all isolates were transferred from the gel to nitro-cellulose membrane, and probed with serum sample obtained from a chronic chagasic patient. Reactions were developed using an anti-human IgG conjugate with alkaline phosphatase and BCIP + NBT solution. Both groups of isolates showed similar reactivity patterns. Immuno-dominant bands were located in the 25-30-kDa region.

This analysis shows more conclusively that although proteins may be from different strains, the same or different proteins may exist and western blot is highly suitable and efficient for this task. Upon analysis and discovery that proteins are obviously different in a western blot then further sequence and mass analyses can be performed. Western blots have the advantage of being rapid, accurate enough to differentiate different band sizes and have a high degree of resolution. All of which are demonstrated in the last investigation.

A parisitology paper entitled "Western Blot Analysis of Excreted/Secreted Antigens from 12 Trypanasoma Cruzi Strains Reveals Conserved and Polymorphic Peptides" was written with the western blot as the central diagnostic tool for the investigation. The excreted/ secreted components from T. cruzi (Y strain) trypomastigotes (TESA) include a 150-160 kDa antigen which displayed high sensitivity and specificity when assayed with Chagas' disease chronic phase sera by Immuno-blotting, whereas acute phase sera showed a characteristic ladder-like neuraminidase/ transialidase pattern (Umezawa et al., J. Clin. Microb.). Here they show that the 150-160 kDa antigen was consistently observed in Immuno-blots of TESA fractions obtained from 12 different T. cruzi strains, indicating that this antigen is highly conserved among strains. On the other hand, the ladder-like reactivity patterns depicted with acute phase sera were extremely polymorphic, since 11 different patterns were obtained with the 12 TESA fractions.

Distinct patterns were also observed when anti-T. Cruzi neuraminidase/transialidase immune serum was incubated with Western-Blots of those fractions, confirming that members of neuraminidase/ transialidase family are the major polymorphic antigens found among the components excreted/secreted by trypomastigotes from different T. cruzi strains.

The high sensitivity of the 150-160 kDa antigen contrasts with the results obtained by Van Voorhis et al. with a FL-160 antigen. Besides, since neither TCA precipitation nor boiling or -20/-70oC storage of TESA fraction interfered with sera reactivity to the 150-160 kDa antigen, this antigen seems also to be different from a heat-labile 160 kDa protein described by Norris et al.

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