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ANTIBODIES
(anti-Human and others as indicated)
RDI Divison of researchd 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.
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.
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 researchd Industries Intl
(Research Diagnostics Inc)
San Jose, 95123 CA Snell ave 658
USA
or 408-780-0908
email: sales@researchd.com
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