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Mouse Cystatin B ELISA Kit

SKU:
MOFI00496
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q62426
Sensitivity:
0.094ng/ml
Range:
0.156-10ng/ml
ELISA Type:
Sandwich
Synonyms:
Cstb, Cystatin B, CSTB, Stefin-B, CPI-B, PME, EPM1, Liver thiol proteinase inhibitor
Reactivity:
Mouse
$779
Frequently bought together:

Description

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Mouse Cystatin B ELISA Kit

The Mouse Cystatin B ELISA Kit is specifically designed for the precise and reliable measurement of cystatin B levels in mouse serum, plasma, and cell culture supernatants. With high sensitivity and specificity, this kit ensures accurate and reproducible results, making it a valuable tool for researchers in various fields.Cystatin B is an important protein with functions in inhibiting cysteine proteases, regulating immune responses, and maintaining cellular homeostasis. Dysregulation of cystatin B has been implicated in various diseases, including cancer, neurodegenerative disorders, and inflammatory conditions.

Therefore, the Mouse Cystatin B ELISA Kit is essential for studying the role of cystatin B in disease progression and developing potential therapeutic strategies.Overall, the Mouse Cystatin B ELISA Kit provides researchers with a reliable and efficient method for quantifying cystatin B levels in mouse samples, contributing to a better understanding of the biological mechanisms involved and facilitating advancements in disease research.

Product Name:Mouse Cystatin B ELISA Kit
Product Code:MOFI00496
Size:96 Assays
Alias:Cstb, Cystatin B, CSTB, Stefin-B, CPI-B, PME, EPM1, Liver thiol proteinase inhibitor
Detection Method:Sandwich ELISA
Application:This immunoassay kit allows for the in vitro quantitative determination of Mouse Cstb concentrations in serum plasma and other biological fluids.
Sensitivity:0.094ng/ml
Range:0.156-10ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery: Matrices listed below were spiked with certain level of Mouse Cstb and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse Cstb in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)88-10498
EDTA plasma(n=5)91-10396
UFH plasma(n=5)85-10093
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse Cstb and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)85-102%85-105%86-104%
EDTA plasma(n=5)85-99%88-97%89-98%
UFH plasma(n=5)82-97%86-99%80-96%
Intra Assay:CV <8%
Inter Assay:CV <10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8-12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

Other materials and equipment required:

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir
UniprotQ62426
UniProt Protein Function:Function: This is an intracellular thiol proteinase inhibitor.
UniProt Protein Details:

Subcellular location: Cytoplasm.

Tissue specificity: Widely expressed. Highest expression in heart, liver and kidney. Lower levels in brain, lung and skeletal muscle. Lowest levels in spleen and testis.

Sequence similarities: Belongs to the cystatin family.

UniProt Code:Q62426
NCBI GenInfo Identifier:6681071
NCBI Gene ID:13014
NCBI Accession:NP_031819.1
UniProt Secondary Accession:Q62426,Q3UAW2,
UniProt Related Accession:Q62426
Molecular Weight:11,046 Da
NCBI Full Name:cystatin-B
NCBI Synonym Full Names:cystatin B
NCBI Official Symbol:Cstb  
NCBI Official Synonym Symbols:Stfb; AA960480  
NCBI Protein Information:cystatin-B; stefin-B
UniProt Protein Name:Cystatin-B
UniProt Synonym Protein Names:Stefin-B
Protein Family:Cystatin
UniProt Gene Name:Cstb  
UniProt Entry Name:CYTB_MOUSE

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

StepProcedure
1. Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.Aliquot 0.1ml standard solutions into the standard wells.
3. Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4. Add 0.1 ml of properly diluted sample (Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37 °C for 90 min.
6. Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7. Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9. Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10. Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11. Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12. Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13. Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type Protocol
Serum:

If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

Plasma:

Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 - g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.

Urine & Cerebrospinal Fluid:

Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.

Cell culture supernatant:

Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.

Cell lysates:

Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20°C.

Tissue homogenates:

The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.

Tissue lysates:

Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.

Breast Milk:

Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

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