Human HSP27 / HSPB1 ELISA Pair Set

Details for Product No. ABIN2010373, Supplier: Log in to see
Antigen
  • zgc:112532
  • HSP27
  • Hs.78846
  • LOH11CR1K
  • MKBP
  • 27kDa
  • 2810021G24Rik
  • heat shock 27kDa protein 2
  • heat shock protein, alpha-crystallin-related, b2
  • heat shock protein 2
  • heat shock protein beta 2
  • HSPB2
  • hspb2
  • LOC100222471
  • LOC100352733
  • Hspb2
Reactivity
Human
Host
Rabbit
Conjugate
HRP
Method Type
Sandwich ELISA
Application
ELISA
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Purpose The Human HSP27 ELISA Pair Set is for the quantitative determination of Human HSP27.
This ELISA Pair Set contains the basic components required for the development of sandwich ELISAs.
Detection Method Colorimetric
Sensitivity 9.4 pg/mL
Characteristics Capture Antibody: 1.0 mg/mL of rabbit anti-HSP27 monoclonal antibody, Dilute to a working concentration of 1 μg/mL in CBS before coating.

Detection Antibody: 0.5 mg/mL mouse anti-HSP27 monoclonal antibody conjugated to horseradish-peroxidase (HRP). Dilute to working concentration of 0.25 μg/mL in detection antibody dilution buffer before use.

Standard: Each vial contains 13 ng of recombinant HSP27. Reconstitute with 1 mL detection antibody dilution buffer. After reconstitution, store at -20 °C to -80 °C in a manual defrost freezer. A seven-point standard curve usi ng 2-fold serial dilutions in sample dilution buffer, and a high standard of 0.6 ng/mL is recommended.

Sensitivity: The minimum detectable dose of Human HSP27 was determined to be approximately 9.4 pg/mL. This is defined as at least three times standard deviations above the mean optical density of 10 replicates of the zero standard.
Components
  • Capture Ab
  • Detection Ab
  • Standard
Material not included CBS - 0.05 M
Na2CO3 - 0.05 M
NaHCO3, pH 9.6, 0.2 μm filtered
TBS - 25 mM
Tris, adjust pH to 7.4 by HCl
Wash Buffer - 0.05 % Tween20 in TBS, pH 7.2 - 7.4
Blocking Buffer - 5 % BSA in Wash Buffer
Sample dilution buffer - 0.5 % BSA in wash buffer, pH 7.2 - 7.4, 0.2 μm filtered
Detection antibody dilution buffer - 0.5 % BSA in wash buffer, pH 7.2 - 7.4, 0.2 μm filtered
Substrate Solution: To achieve best assay results, fresh substrate solution is recommended Substrate stock solution - 10 mg/mL TMB (Tetramethylbenzidine ) in DMSO Substrate dilution buffer - 0.05 M Na2HPO4 and 0.025 M citric acid , adjust pH to 5.5
Substrate working solution - For each plate dilute 250 μL substrate stock solution in 25 mL substrate dilutionbuffer and then add 80 μL 0.75 % H2O2 , mix it well
Stop Solution - 2 N H2SO4
Alternative Name HSPB2
Background Heat shock proteins (HSPs) are a group of highly conserved stress response proteins which primarily function asmolecular chaperones, and are involved in a variety of biological processes including thermotolerance, inhibition ofapoptosis, regulation of cell development and cell differentiation, as well as signal transduction. As a member of thechaperones, HSP27 appears in many cell types, especially all types of muscle cells, and is suggested to play anessential role in the differentiation of tissues. HSP27 has a characteristic and highly conserved amino acid sequence atthe C-terminus, the so-called α-crystallin-domain which is important for the formation of stable dimers, and it isdemonstrated that the oligomerization status is connected with the chaperone activity. HSP27 is also identified as ananti-apoptotic molecule. It interacts with the outer mitochondrial membranes and interferes with the activation ofcytochrome c/Apaf-1/dATP complex and therefore inhibits the activation of procaspase-9 and procaspase-3. Furthermore, HSP27 also exerts functions in protecting actin filaments from fragmentation and the activation of thecertain proteasome.
Research Area Signaling, Heat Shock Proteins
Application Notes Optimal working dilution should be determined by the investigator.
Protocol This ELISA Pair Set is a solid phase sandwich ELISA (Enzyme-Linked Immunosorbent Assay). It utilizes a monoclonal antibody specific for HSP27 coated on a 96-well plate. Standards and samples are added to the wells, and any HSP27 present binds to the immobilized antibody. The wells are washed and a horseradish peroxidase conjugated mouse anti-HSP27 monoclonal antibody is then added, producing an antibody-antigen-antibody "sandwich". The wells are again washed and TMB substrate solution is loaded, which produces color in proportion to the amount of HSP27 present in the sample. To end the enzyme reaction, the stop solution is added and absorbances of the microwell are read at 450 nm.
Reagent Preparation
  1. Dilute the capture antibody to the working concentration in CBS. Immediately coat a 96-well microplate with 100 μL per well of the diluted capture antibody. Seal the plate and incubate overnight at 4 °C.
    2. Aspirate each well and wash with at least 300 μL wash buffer, repeating the process two times for a total of three washes. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining wash buffer by inverting the plate and blotting it against clean paper towels.
    3. Block plates by adding 300 μL of blocking buffer 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.
Assay Procedure
  1. Add 100 μL of sample or standards in sample dilution buffer per well. Seal the plate and incubate 2 hours at roomtemperature.
    2. Repeat the aspiration/wash as in step 2 of plate preparation.
    3. Add 100 μL of the detection antibody, diluted in antibody dilution buffer, to each well. Seal the plate and incubate 1hour at room temperature.
    4. Repeat the aspiration/wash as in step 2 of plate preparation.
    5. Add 200 μL of substrate solution to each well. Incubate for 20 minutes at room temperature (if substrate solutionis not as requested, the incubation time should be optimized). Avoid placing the plate in direct light.
    6. Add 50 μL of stop solution to each well. Gently tap the plate to ensure thorough mixing.
    7. Determine the optical density of each well immediately, using a microplate reader set to 450 nm.
Calculation of Results

Calculate the mean absorbance for each set of duplicate standards, controls and samples. Subtract the mean zero standard absorbance from each. Construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. To determine the concentration of the unknowns, find the unknowns' mean absorbance value on the y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the x-axis and read the concentration. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. Alternatively, computer-based curve-fitting statistical software may also be employed to calculate the concentration of the sample.

Restrictions For Research Use only
Format Liquid
Handling Advice Avoid repeated freeze-thaw cycles.
Storage -20 °C,-80 °C
Storage Comment Capture Antibody: Aliquot and store at -20°C to -80°C for up to 6 months from date of receipt. Avoid repeated freeze-thaw cycles.

Detection Antibody: Protect it from prolonged exposure to light. Aliquot and store at -20°C to -80°C and for up to 6 months from date of receipt. Avoid repeated freeze-thaw cycles.

Standard: Store lyophilized standard at -20°C to -80°C for up to 6 months from date of receipt. Aliquot and store the reconstituted Standard at -80°C for up to 1 month. Avoid repeated freeze-thaw cycles.
Expiry Date 6 months
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