The Human CLPP (Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit) ELISA Kit is a powerful tool for the precise measurement of CLPP levels in human samples including serum, plasma, and cell culture supernatants. With exceptional sensitivity and specificity, this kit delivers dependable and consistent results, making it an invaluable asset for various research endeavors.CLPP is a critical component of mitochondrial protein quality control, playing a crucial role in maintaining mitochondrial homeostasis and cell function.
Dysregulation of CLPP has been linked to various diseases such as neurodegenerative disorders, metabolic syndromes, and aging-related conditions, highlighting its significance as a potential target for therapeutic interventions.Equip yourself with the Human CLPP ELISA Kit to delve deeper into the intricate mechanisms of mitochondrial biology and explore novel therapeutic strategies for a wide array of diseases and disorders.
Product Name:
Human CLPP ELISA Kit
Product Code:
HUFI01889
Size:
96 Assays
Alias:
CLPP, Endopeptidase Clp
Detection method:
Competitive ELISA, Coated with Antibody
Application:
This immunoassay kit allows for the in vitro quantitative determination of Human CLPP concentrations in serum plasma and other biological fluids.
Sensitivity:
0.938ng/ml
Range:
1.56-100ng/ml
Storage:
4°C for 6 months
Note:
For Research Use Only
Recovery:
Matrices listed below were spiked with certain level of Human CLPP and the recovery rates were calculated by comparing the measured value to the expected amount of Human CLPP in samples.
Matrix
Recovery range(%)
Average(%)
serum(n=5)
87-104
94
EDTA plasma(n=5)
89-105
96
UFH plasma(n=5)
94-105
100
Linearity:
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CLPP and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample
1:2
1:4
1:8
serum(n=5)
95-105%
89-93%
85-101%
EDTA plasma(n=5)
82-101%
86-99%
83-100%
UFH plasma(n=5)
81-90%
83-95%
83-100%
CV(%):
Intra-Assay: CV<8% Inter-Assay: CV<10%
Component
Quantity
Storage
ELISA Microplate(Dismountable)
8×12 strips
4°C for 6 months
Lyophilized Standard
2
4°C/-20°C
Sample/Standard Dilution Buffer
20ml
4°C
Biotin-labeled Antibody(Concentrated)
60ul
4°C (Protect from light)
Antibody Dilution Buffer
10ml
4°C
HRP-Streptavidin Conjugate(SABC)
120ul
4°C (Protect from light)
SABC Dilution Buffer
10ml
4°C
TMB Substrate
10ml
4°C (Protect from light)
Stop Solution
10ml
4°C
Wash Buffer(25X)
30ml
4°C
Plate Sealer
5
-
Other materials and equipment required:
Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipettetips
Protease component of the Clp complex that cleaves peptides and various proteins in an ATP-dependent process. Has low peptidase activity in the absence of CLPX. The Clp complex can degrade CSN1S1, CSN2 and CSN3, as well as synthetic peptides (in vitro) and may be responsible for a fairly general and central housekeeping function rather than for the degradation of specific substrates.
NCBI Summary:
The protein encoded by this gene belongs to the peptidase family S14 and hydrolyzes proteins into small peptides in the presence of ATP and magnesium. The protein is transported into mitochondrial matrix and is associated with the inner mitochondrial membrane. [provided by RefSeq, Jul 2008]
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Equilibrate the TMB substrate for at least 30 min at 37°C beforeuse. When diluting samples and reagents, they must be mixed completely andevenly. It is recommended to plot a standard curve for each test.
Step
Protocol
1.
Set standard, test sample and control (zero) wells on the pre-coatedplate respectively, and then, record their positions. It isrecommended to measure each standard and sample in duplicate. Washplate 2 times before adding standard, sample and control (zero) wells!
2.
Add Sample and Biotin-detection antibody: Add 50µL of Standard, Blank or Sample per well. The blankwell is added with Sample Dilution Buffer. Immediately add 50 µL of biotin-labelled antibody workingsolution to each well. Cover with the plate sealer provided. Gently tap the plate to ensure thoroughmixing. Incubate for 45 minutes at 37°C. (Solutions are added to the bottom of micro-ELISA platewell, avoid touching plate walls and foaming).
3.
Wash: Aspirate each well and wash, repeating the process three timesWash by filling each well with Wash Buffer (approximately 350µL)using a squirt bottle, multi-channel pipette, manifold dispenser orautomated washer. Complete removal of liquid at each step is essentialto good performance. After the last wash, remove any remaining WashBuffer by aspirating or decanting. Invert the plate and pat it againstthick clean absorbent paper.
4.
HRP-Streptavidin Conjugate(SABC): Add 100µL of SABC workingsolution to each well. Cover with a new Plate sealer. Incubate for30minutes at 37°C.
5.
Wash: Repeat the aspiration/wash process for five times.
6.
TMB Substrate: Add 90µL of TMB Substrate to each well. Coverwith a new Plate sealer. Incubate for about 10-20 minutes at 37°C.Protect from light. The reaction time can be shortened or extendedaccording to the actual color change, but not more than 30minutes.When apparent gradient appeared in standard wells, you can terminatethe reaction.
7.
Stop: Add 50µL of Stop Solution to each well. Color turn toyellow immediately. The adding order of stop solution should be as thesame as the substrate solution.
8.
OD Measurement: Determine the optical density (OD Value) of each wellat once, using a microplate reader set to 450 nm. You should open themicroplate reader ahead, preheat the instrument, and set the testing parameters.
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.
