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ORDERING INFORMATION
Catalog Number: MAB726
Clone: 22210
Lot Number: AYF04
Size: 500 µg
Formulation: 0.2 µm filtered solution in PBS
with 5% trehalose
Storage: -20o
 C
Reconstitution: sterile PBS
Specificity: human TNF RII
extracellular domain
Immunogen: E. coli-derived rhTNF RII
extracellular domain
Ig class: mouse IgG1
Applications: ELISA capture
Neutralization of bioactivity
Western blot

Monoclonal
Anti-human TNF RII/TNFRSF1B Antibody
Preparation
This antibody was produced from a hybridoma resulting from the fusion of a mouse
myeloma with B cells obtained from a mouse immunized with purified, E. coli-derived,
ecombinant human tumor necrosis factor receptor type II （rhTNF RII） extracellular domain.
The IgG fraction of the tissue culture supernatant was purified by Protein G affinity
chromatography. TNF RII, also known as p75 or p80 TNF R, is a prototypic member of the
TNF receptor superfamily and has been designated TNFRSF1B. It binds TNF with high
affinity and is expressed primarily in cells of the immune system.
Formulation
Lyophilized from a 0.2 µm filtered solution in phosphate-buffered saline （PBS） with 5%
rehalose.
Reconstitution
Reconstitute with sterile PBS. If 1 mL of PBS is used, the antibody concentration will be
500 µg/mL.
Storage
Lyophilized samples are stable for twelve months from date of receipt when stored
at -20° C to -70° C. Upon reconstitution, the antibody can be stored at 2° - 8° C for 1 month
without detectable loss of activity. Reconstituted antibody can also be aliquotted and stored
rozen at -20° C to -70° C in a manual defrost freezer for six months without detectable
oss of activity. Avoid repeated freeze-thaw cycles.
Specificity
This antibody was selected for use as a capture antibody in human TNF RII sandwich
ELISAs.Applications
Neutralization of Human TNF RII Bioactivity - The exact concentration of antibody
required to neutralize human TNF RII activity is dependent on the cytokine concentration,
cell type, growth conditions and the type of activity studied. To provide a guideline, R&D
Systems has determined the neutralization dose for this antibody under a specific set of
conditions. The Neutralization Dose50 （ND50） for this antibody is defined as that
concentration of antibody required to yield one-half maximal inhibition of the cytokine
activity on a responsive cell line, when that cytokine is present at a concentration just high
enough to elicit a maximum response.
The ND50 for this lot of anti-human TNF RII antibody was determined to be approximay
0.5 - 1.5 µg/mL in the presence of 0.3 µg/mL of rhTNF RII and 0.25 ng/mL of rhTNF-α,
using the mouse L929 cell line as an assay. The specific conditions are described in the
figure legends.Western Blot - This antibody can be used at 1 - 2 µg/mL with the appropriate secondary
reagents to detect human TNF RII. The detection limit for rhTNF RII is approximay
0.5 ng/lane under non-reducing conditions. In western blots, this antibody does not
cross-react with rhTNF RI, rmTNF RI or rmTNF RII.
ELISA Capture - This antibody can be used as a capture antibody in a human TNF RII
ELISA in combination with biotinylated, human TNF RII affinity purified polyclonal detection
antibody （Catalog # BAF726）. A general protocol is provided on the next page. Using plates
coated with 100 µL/well of the capture antibody at 2 µg/mL, in combination with 100 µL/well
of the detection antibody, an ELISA for sample volumes of 100 µL can be obtained. To
arrive at the optimal dose range for this ELISA, set up a two-fold dilution series of the
protein standard starting with 0.5 ng/mL. In this format, no cross-reactivity was observed
with rhTNF RI/TNFRSF1A, rmTNF RI/TNFRSF1A, rmTNF RII/TNFRSF1B,
rmTNF-α/TNFSF1A, rhTNF-α/TNFSF1A or rhTNF-β/TNFSF1B.
Optimal dilutions should be determined by each laboratory for each application.
Figure 1: Human TNF RII inhibits the TNF-induced mouse L-929 cell lysis in a dose-
dependent manner. The ED50 for this effect is typically 0.05 - 0.1 µg/mL, in the presence of
0.25 ng/mL rhTNF-α and 1 µg/mL of actinomycin D.
Figure 2: Approximay 0.5 - 1.5 µg/mL of the antibody will neutralize 50% of the bioactivity
due to 0.3 µg/mL of rhTNF RII and 0.25 ng/mL of rhTNF-α.ELISA Protocol
Solutions Required
•  Wash Buffer - 0.05% Tween 20 in PBS, pH 7.4
•  Diluent - 1 % BSA in Phosphate-buffered Saline pH 7.4.
•  Substrate Solution - 1:1 mixture of Color Reagent A （H202） and Color Reagent B （Tetramethylbenzidine） （R&D Systems,
Catalog # DY999）
•  Stop Solution - 1 M H2SO4
Plate Preparation
1.  Transfer 100 µL/well of the capture antibody （diluted to the appropriate concentration in PBS, use immediay） to an ELISA plate. Seal
plate and incubate overnight at room temperature.
2.  Aspirate each well and wash with Wash buffer, repeating the process two times for a total of 3 washes.  Wash by filling each well with
Wash Buffer （400 µL） using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher.  Complete removal of liquid at each
step is essential to good performance.  After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and
blotting it against clean paper toweling.
3.  Block plates by adding 300 µL of PBS containing 1% BSA, 5% sucrose and 0.05% NaN3 to each well. Incubate at room temperature for a
minimum of 1 hour.
4.  Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition. Alternatively, the blocking buffer can be aspirated
after step 3 and the plates can be dried under vacuum. When sealed with desiccant, the plates can be stored at 4° - 8° C for at least
2 months.Assay Procedure
1.  Dilutions of unknowns and standards should be carried out in polypropylene tubes. Add 100 µL of sample or standards in an appropriate
diluent, per well.  Mix by gently tapping the plate frame for 1 minute.  Cover with an adhesive strip and incubate 2 hours at room
temperature.
2.  Repeat the aspiration/wash as in step 2 of Plate Preparation.
3.  Dilute the biotinylated detection antibody with 2% heat-inactivated normal goat serum in the above diluent and mix gently for 1 hour.
Add 100 µL to each well. Cover with a new adhesive strip and incubate for 2 hours at room temperature.
4.  Repeat the aspiration/wash as in step 2 of Plate Preparation.
5.  Add 100 µL streptavidin HRP （R&D Systems, Catalog # DY998, dilute according to the directions on the vial label） to each well. Cover the
plate and incubate for 20 minutes at room temperature.
6.  Repeat the aspiration/wash as in step 2.
7.  Add 100 µL of Substrate Solution to each well. Incubate for 20 - 30 minutes at room temperature. Avoid placing the plate in direct light.
8.  Add 50 µL of Stop Solution to each well.  Gently tap the plate to ensure thorough mixing.
9.  Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.  If wavelength correction is
available, set to 540 nm or 570 nm.  If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at
450 nm.  This subtraction will correct for optical imperfections in the plate.  Readings made directly at 450 nm without correction may be
higher and less accurate.Calculation of Results
To calculate assay results, average the duplicate readings and subtract the zero standard optical density from the sample optical density.
Create a standard curve using data reduction software capable of generating a four parameter （4P-L） curve fit. Alternatively, plot the optical
density for the standards versus the concentration of the standards and draw the best curve. The data can be linearized by using log-log paper
and regression analysis may be applied to the log transformation. To determine the human TNF RII concentrations for each sample, first find
the absorbance value on the y-axis and extend a horizontal line to the standard curve. At the point of intersection, extend a vertical line to the
x-axis and read the corresponding human TNF RII concentration. If samples have been diluted, the concentration read from the standard curve
must be multiplied by the dilution factor. A standard curve should be generated for each set of samples assayed.Limitations
It is important that the diluents selected for reconstitution and for dilution of the standard reflect the environment of the samples being
measured. The diluent suggested in the above protocol may be suitable for most cell culture supernate samples. Validate diluents for specific
sample types prior to use.
A basic understanding of immunoassay development is required for the successful use of these reagents in immunoassays. The protocol
provided is for demonstration purposes only. The type of enzyme and substrate and the concentrations of capture/detection antibodies used
can be varied to create an immunoassay with a different sensitivity and dynamic range.