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ANTIBODIES  

(anti-Human and others as indicated)

RDI Divison of Fitzgerald Industries Intl  offers a wide line of  antibodies. Since no one antibody works best for all applications (neutralization, blotting, histochemistry, ELISA, etc), we offer many different types of antibodies to help solve this problem. Please inquire for other applications or types of antibodies not listed below.

Update January 13, 1998, :Note clones  1B2 & 2C5 (clone 2C5 also suitable for paraffin sections/histochemistry)

-see also new Goat anti-ubiquitin L1 antibody

Catalog Number:  RDI-ALS24509    $344.00/100ul

Clone: Ubi-1

Quantity: 100ul

Description: Ubiquitin is a highly conserved globular 76 amino acid protein of about 8.5 kDa molecular weight. It has a important role in the targeting of proteins for proteolytic degradation. Proteins to be degraded are covalantly coupled to the C-terminus of ubiquitin by means of ubiquitin ligases. The ubiquitin itself is frequently also ubiquitinated, producing a polyubiquitin chain. The polyubquitinated complex is then recognized by a complex of degradative enzymes which together form the proteosome. Interestingly, ubiquitin also becomes covalently bonded to many types of pathological inclusions seen in serious human disease states which appear to be resistant to normal degradation, so that ubiquitin antibodies are very useful for studies of these inclusions. For example the neurofibrillary tangles and paired helical filaments diagnostic of Alzheimer's disease, the Lewy bodies seen in Parkinson's disease, and Pick bodies found in Pick's disease are all heavily ubiquitinated and can all be readily visualized with ubiquitin antibodies of appropriate specificity.

Host Species: Mouse

Specificity: Ubi-1 was raised against purified ubiquitin conjugated with glutaraldehyde to keyhole limpet hemocyanin. The clone was initially screened on ELISA of the immunogen, and subsequently tested on sections of Alzheimer brain. Ubi-1 was one of several clones which stained neurofibrillary tangles in frozen sectioned material strongly and specifically. Subsequent studies indicated that MCA-Ubi-1 is relatively insensitive to formalin fixation and so can be used on mildly fixed histological sections of human brain for studies of Alzheimer's disease. The antibody also works on paraffin embedded material. Ubi-1 also recognizes other ubiquitinated inclusion bodies such as the Lewy bodies of Parkinson's disease and the Pick bodies in Pick's disease in formalin fixed tissues. Ubi-1 will recognize human, bovine, chicken, Drosophila, and C. elegans in ELISA. Ubi-1 also works on Western blots and can be used to study ubiquitinated proteins. It has also been successfully used in competitive ELISA.

Applications: Ubi-1 is excellent for the detection of ubiquitinated inclusions seen in human neurodegenerative diseases such as the neurofibrillary tangles of Alzheimer's disease. We recommend a starting dilution of 1:1,000 for this purpose, using ABC or other enzymatic amplification procedures. For immunofluorescence, use Ubi-1 diluted 1:500. For western blots use Ubi-1 diluted 1:1,000.

Format: Sterile cell culture fluid plus sodium azide. Ubi-1 is a mouse IgG1 with a kappa light chain. Supplied at a concentration of 1mg/ml.

Storage: Maintain at +2-8°C for 3 months or at -20°C for longer periods. Stable for 1 year. Avoid repeated freeze-thaw cycles.

References: Boutajangout A, Authelet M, Blanchard V, Touchet N, Tremp G, Pradier L, Brion JP. Neurobiol Dis. 15:47-60 (2004).

Wang DS, Bennett DA, Mufson EJ, Mattila P, Cochran E, Dickson DW. Neurosci. Res. 48:93-100 (2004).

He CZ, Hays AP. J. Neurol. Sci. 217:47-54 (2004).

For research use only; not for use as a diagnostic.

Background: Ubiquitin protein conjugates associated with filamentous inclusions or in dot like strcutures (lyosome-releated organelles) are characteristic of the major human idiopathic neurodegenerative diseases. Dot-like strcutures are aslo a feature of the transmissable neurodegenerative diseases. This antibody detects neurofibrillary TANGLES in Alzheimer's disease, Lewy bodies in Lewy Body disease, Rosenthal fibres in astrocytomas, Mallory bodies in Alcoholic Liver Disease. Also detects dot-like structures in the white matter of normal aging brain.

Catalog#: RDI-UBIQUITabm   Price: $438.00/vial

Package Size: 1ml lyophilized (tissue culture supernatant containing 15mM sodium azide). Reconstitute with 1ml distilled water.

Clone: FPM1

Isotype: mouse IgG1
Hybridoma Partner: mouse myeloma (p3-NS1-Ag4-1)

Antigen: ubiquitin conjugated with glutaraldehyde cross linked to keyhole limpet haemocyanin.

Activity: Positive staining can be observed in brain sections from patients with Alzheimer's disease. Stains Cytoplasmic filamentous inclusions and dot-like immunoreactivity.

Application
-paraffin wax embedded tissue(1:25-1:50 dilution) Incubate 60 minutes at room temp, visualize with standard ABC techniques.

-western blotting

Storage: Store at 4 Deg C or below. Store reconstituted material in aliquots at -20 DEG C. Avoid frequent freeze thaw cycles.

general ref: -Lennox G, Lowe J et al, Journal of Neurosurgery and Psychiatry 52:1236- 2347(1989) and 52:67-71(1989)

Precautions:For In vitro research Use Only. Not for use in or on humans or animals or for diagnostic It is the responsibility of the user to comply with all local/state and Federal rules in the use of this product. We are not responsible for any patent infringements that might result with the use of or derivation of this product.

Code No.               Clone       Form        Quantity                Presentation

RDI-UBIQ-1B3      1B3         Purified      100ul (1mg/ml)    Liquid

RDI-UBIQ-2C5      2C5        Purified       100ul (1mg/ml)    Liquid  (also for histochemistry)

Immunogen Purified bovine erythrocyte ubiquitin

Hybridoma Myeloma P3 U1 X P1 mouse (C57BL/6xBalb/c) spleen cells

Isotype IgG1

Specificity: The antibodies react with human erythrocyte ubiquitin and bovine erythrocyte ubiquitin. These antibodies (1B3 and 2C5) recognize different epitope sites each other.

Presentation:Purified from ascites fluid by ammonium sulfate precipitation and affinity chromatography on protein A sepharose. Sodium azide (0.1%) has been added as preservative.

Western immunoblotting was performed as follows.

3 ug of ubiquitin (From bovine red blood cell-) or 20ul of WIL 2 cell extracts (10(8) cells were suspended with 5 ml of SDS PAGE sample buffer and sonicated 30 sec) were first applied to a 10% polyacrylamide gel and subjected to electrophoresis according to the method of Laemmli, except for substituting the Glysinc cathode buffer with the Tricine cathode buffer (0.1M Tricine; no correction of the pH, which is around 8.25). After electrophoresis, the protein bands were transferred onto nitrocellulose paper using a Bio-Rad transblotting unit of 25 mM Tris, 192 mM glycine, 20% methanol. The nitrocellulose paper was incubated 2 h with 0.2% skim milk in PBS at room temperature. The paper was then incubated with 1ug/ml of anti ubiquitin antibodies (clone 1B3 or -2C5) in 0.2% skim milk in PBS for 2h at room temperature followed by three 5-min washes in PBS. The paper was then incubated with peroxidase conjugated rabbit anti-mouse Ig 1:125 dilution in 1% BSA in PBS) for 45 min at room temperature. After another three 5-min washes in PBS, the paper was developed with a freshly prepared solution of 3,3'-diaminobenzidine tetrahydrochloride (25 ug/ml in PBS,0.015% hydrogen peroxide).

PROTOCOL

Flow Cytometric Analysis of anti ubiquitin I

RDI-UBIQ-1B3    &    RDI-UBIQ-2C5

INDIRECT LABELLING ON WHOLE CELL WITH PURIFIED FORM

1. 10(6) to 2 X 10(6) cells are washed 3 times with PBS(-) containing 2% FCS,0.1% NaN3.

2. 200ul of the fixing reagent (4% PFA ub 0.1M NaH(2)PO(4)(pH 7.4)) is added and vortexed immediately. The mixture is in cubated for 30 min. at room temperature.  -OR (70% ETOH -20 DEG C 30min)

  -Note: in fluorescent microscopy, clone 2C5 stained positive only with PFA fixation (clone 1B3 was not reactive).

3. After fixation, 1ml of PBS(-) containing 2% FCS, 0.1% NaN3 are added, followed by centrifugation. The supernatant is discarded and 1 ml of 0.5% Tween 20 are added and incubated for 30 min at room temperature.

4. Washed with PBS(-) containing 2% FCS, 0.1% NaN3.

5. 10ul of normal goat serum are added and incubated for 10 min at room temperature.

6. 30ul of anti ubiquitin (1B3 or 2C5 10ug/ml) are added and incubated for 30 min. at room temperature.

7. Washed twice with PBS(-) containing 2% FCS, 0.1% NaN3.

8. To the cell pettet 20 ul of anti mouse IgG (H+L)-FITC are added and incubated for 30 min. at room temperature.

9. Washed twice with PBS containing 2% FCS, 0.1% NaN3.

10.The pellet is taken up in 500ul of PBS(-) containing 2% FCS, 0.1% NaN3. The cells are now ready for analysis.

FLOW CYTOMETRIC ANALYSIS OF anti ubiquitin II

RDI-UBIQ-1B3 (1B3)    &     RDI-UBIQ-2C5        Raji cell

INDIRECT LABELLING ON WHOLE CELL WITH PURIFIED FORM

1. 10' 2X 106 cells are washed 3 times with PBS(-) containing 2% FCS, 0.1% NaN3.

2. 200ul of the fixing reagent (4% PFA in 0,1 M NaH2PO4 (pH 7.4) is added and vortexed immediately. The mixture is incubated for 30 min. at room temperature.

3. After fixation, 1ml of PBS containing 2% FCS, 0.1% NaN3 are added, followed by centrifugation. The supernatant is discarded and 1 ml of 0.5% Tween 20 are added and incubated for 30 min. at room temperature.

4. Washed with PBS containing 2% FCS, 0.1% NaN3.

5. 10ul of normal goat serum are added and incubated for 10 min at room temperature.

6. 30ul of anti ubiquitin (1B3 or 2C5 10ug/ml) are added and incubated for 30 min. at room temperature.

7. Washed twice with PBS containing 2% FCS, 0.1% NaN3.

8. To the cell pellet 20ul of anti mouse IgG -FITC are added and incubated for 30 min. at room temperature.

9. Washed twice with PBS containing 2% FCS, 0.1% NaN3.

10.The pellet is taken up in 500ul of PBS containing 2% FCS, 0.1% NaN3. The cells are now ready for analysis.

For In Vitro Research Use Only

RDi Div of Fitzgerald Industries Intl

(Research Diagnostics Inc)

34 Junction Square Drive

Concord MA 01742-3049

USA

phone (978) 371-6446 or (800) 370-2222

fax     (978) 371-2266

email: sales@researchd.com

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