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p53 (human) ELISA Kit

Sensitive, Colorimetric Assay
Catalog #: K4829
$525.00

Product Details

Cat # +Size K4829-100
Size 100 assays
Detection Method Absorbance (450 nm)
Species Reactivity human
Applications This ELISA kit is used for quantitative measurement of native and recombinant p53
Features & Benefits • Easy, convenient and time-saving method to assay for human p53.
• Detection Range: 0.2 ng/ml to 25.6 ng/ml.
• This ELISA kit shows no cross-reactivity with other relevant proteins.
• Cross Reactivity: No significant cross-reactivity or interference between this analyte and its analogues was observed.
Kit Components • Plate Coated with p53 Ab
• Assay Diluent
• Wash Buffer A (10x)
• Wash Buffer B (10x)
• Wash Buffer C (10x)
• rh p53 Standard
• Detection Antibody (100x)
• Streptavidin-HRP Conjugate (100x)
• TMB Substrate
• Stop Solution
• Plate Sealer
Storage Conditions Multiple Temperatures
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.

Details

BioVision’s p53 Enzyme-Linked Immunosorbent Assay (ELISA) Kit is an in vitro assay for the quantitative measurement of native and recombinant human p53. The p53 protein is encoded by the TP53 gene. It is composed of 393 amino acids but its apparent molecular mass is 53 kDa. p53 binds to a DNA consensus sequence, the p53 response element, and regulates normal cell cycle events by activating transcription of genes involved either in progression through the cell cycle, or causing arrest in G1 when the genome is damaged. It acts as a pivotal suppressor of inappropriate cell proliferation. By initiating suppressive effects through induction of apoptosis, cell senescence, or transient cell-cycle arrest, p53 plays an important role in cancer suppression, developmental regulation, and aging. p53 is found in increased amounts in a wide variety of transformed and tumor cells, where its concentration is increased 5-1000 fold over the concentration in normal cells. Meanwhile, p53 is also frequently mutated or inactivated in about 60% of cancers. The p53 ELISA assay employs an antibody specific for human p53 coated on a 96-well plate. Standards and samples are pipetted into wells and p53 present in the sample is bound to the wells by immobilized antibody. Wells are washed and a biotin-labeled human p53 specific detection antibody is added. After washing away unbound detection antibody, a streptavidin HRP-conjugate is added to the wells. The wells are again washed, a TMB substrate solution is added, and color develops in proportion to the amount of bound p53. Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. Sensitivity of the kit is 0.1 ng/ml and detection range is from 0.2 ng/ml to 25.6 ng/ml. Recovery within this range is 86.3%. The intra-assay reproducibility as measured by the coefficient of variation (CV) is < 8 % & inter-assay has CV < 12 %.


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What is a sandwich ELISA?
The analyte of interest in a sample is "sandwiched" by an immobilized capture antibody coated on the plate and a labeled antibody used for detection. Sandwich ELISA is very sensitive. The density of color is proportional to the amount of analyte captured from the samples and can be quantified when compared with standard curve.
What is a competitive ELISA?
The endogenous unlabled analyte of interest in a sample is "competed" by the exogenous labeled antigen coated on the plate for a limited amount of antibody binding sites. Therefore, the lower signal indicate higher concentration of the analyte.
What dilution range should I use for the samples?
A preliminary experiment is always recommended when working with new samples and ELISA Kits to ensure all results fall within the detection range.
Can I extend the standard curve (in either direction)?
Extended standard curve is not recommended by BioVision.
What type of software is needed to graph a 4-parameter or 5-parameter curve?
SoftMax Pro by Molecular Devices, SigmaPlot® by Systat Software Inc., or others can be used for this purpose.
Low absorbance; No signal
Target present below detection limits of assay
Reduce dilution factor to increase sample concentration  (Preliminary experiment is recommended for optimal dilution range)
Incompatible sample types
Use samples recommended by the kit
Standard dilutions are unstable
Use fresh standard dilutions
Buffers/substrates are contaminated
Use new buffers and substrates
Insufficient incubation time 
Extend incubation time
Cover or seal plates during all incubation 
Plate washings too vigorous
Pipette wash buffer gently or make sure the automatic wash system has correct pressure
Wells scratched with pipette or washing tips
Use caution when dispensing and aspirating into and out of wells
Incubation temperature too low
Ensure incubations are carried out at temperatures recommended by the manual
Reagents are cold
Bring all reagents to room temperature before each assay
Plate read at incorrect wavelength

Ensure the plate reader is set at the correct wavelength recommended on the protocol

High Background
Insufficient washing
Increase number of washes; Add a 30 second soaking step in between washes;  at the end of each washing step, invert plate on absorbent tissue to completely drain all wells
Residual buffer/substrates remain in wells
Remove all residual buffer and substrates at the end of each wash
Too much HRP-Streptavidin
Centrifuge and mix the substrate vial before use to avoid percipitation and inconsistent HRP concentration
Excessive incubation time
Reduce incubation time
Substrate incubation carry out in light or wait too long to read plate after adding stop solution
Avoid light during incubation and read signals immediately after adding stop solution
Standards improperly reconstituted or diluted
Briefly spin vials before opening; inspect for undissolved material after reconstitution
Poor standard curve
Standard improperly diluted
Confirm dilutions are made correctly
Standard dilutions are unstable
Use freshly diluted standards
Pipetting error 
use calibrated pipette and stay consistent between each pipette
Curve doesn't fit scale
Try plotting using different scales  (log-log, 4 parameter logistic, 5 parameter etc.)
Large intra-coefficient of variation (CV)
Bubbles in wells
Avoid bubbles during experiment, eliminate all bubbles prior to reading
Insufficient washing
Increase number of washes; add a 30 second soak step in between washes;  at the end of each washing step, invert plate on absorbent tissue to completely drain all wells
Wells not washed equally/thoroughly
Use calibrated (automated) multi-channel pipette for consistent handling, make sure all parts of the automated washer are unobstructed
Edge effects
Seal the plate completely during incubation
Incomplete reagent mixing
Make sure all reagents are mixed thoroughly during each step
Inconsistent sample preparation or storage
Consistent sample preparation. Optimize sample storage conditions and minimize freeze and thaw cycles
Stacked plates
Avoid stacking plates during incubation
Plate sealers not used or reused
Cover assay plate with plate sealer, use a fresh sealer each time to prevent contamination
Large inter-coefficient of variation (CV))
Insufficient washing
Increase number of washes; add a 30 second soak step in between washes;  at the end of each washing step, invert plate on absorbent tissue to completely drain all wells
Inconsistent incubation temperature
Make sure to follow recommended incubation temperatures. Avoid fluctuations in temperature due to environmental conditions.
Plate sealers not used or reused
Cover assay plate with plate sealer, use a fresh sealer each time to prevent contamination