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ANTIBODIES
(anti-Human and others as indicated)
RDI Division 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.
4clones available for western blotting, flow cytometry and histochemistry use
Update January 13, 1998, :Note clones 1B2 & 2C5 (clone 2C5 also suitable for paraffin sections/histochemistry)
-see also new Goat anti-ubiquitin L1 antibody
DATA SHEET: Mouse anti-Ubiquitin monoclonal
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.
MONOCLONAL ANTIBODY: UBIQUITIN $500.00/vial
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.
The antibody can detect a ubiquitin band migrated to molecular weight of 5.5 Kd by SDS-PAGE.
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)
-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.
PROTOCOL
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)
phone (978) 371-6446 or (800) 370-2222
fax (978) 371-2266
email: sales@researchd.com
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