Human Caspase 4 ELISA Kit (HUFI02292)- High Sensitivity

Description

Description

Human Caspase 4 ELISA

Caspase 4 encodes a protein that plays a central role in the execution-phase of cell apoptosis. Activation of Caspase 4 requires proteolytic processing at conserved internal aspartic residues to generate a heterodimeric enzyme consisting of the large and small subunits. Diseases associated with Caspace 4 include Cystinosis and Neuroblastoma. The Assay Genie Human Caspase 4 ELISA is a highly sensitive assay for the quantitative measurement of Caspase 4 in serum, blood, plasma, cell culture supernatant and tissue samples.

Datasheet MSDS

Key Features

	Save Time	Pre-coated 96 well plate
	Quick Start	Kit includes all necessary reagents
	Publication Ready	Reproducible and reliable results

Overview


Product Name:	Human Caspase 4 ELISA Kit
Product Code:	HUFI02292
Size:	96 Assays
Alias:	CASP4, ICE, relII, ICH-2, Mih1, TX, TX, apoptotic cysteine protease Mih1, TX, CASP-4, caspase 4, apoptosis-related cysteine peptidase, caspase 4, apoptosis-related cysteine protease, caspase-4, ICE, relII, ICE, rel-II, ICEREL-II, ICH2, ICH-2, Mih1, TX, Protease ICH-2, Protease TX, TX
Detection Method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human CASP4 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

Additional Information

Recovery:

Matrices listed below were spiked with certain level of Human CASP4 and the recovery rates were calculated by comparing the measured value to the expected amount of Human CASP4 in samples.


Matrix	Recovery Range (%)	Average (%)
serum (n=5)	85-104	98
EDTA plasma (n=5)	88-105	94
UFH plasma (n=5)	87-105	96

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CASP4 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)	89-105%	89-105%	85-102%
EDTA plasma (n=5)	83-93%	86-101%	82-99%
UFH Plasma (n=5)	85-100%	80-95%	83-99%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Kit Components

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8x12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/ -20°C
Sample/Standard Dlution Buffer	20ml	4°C
Biotin-labeled Antibody (Concentrated)	120ul	4°C (Protection 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 (Protection from light)
Stop Solution	10ml	4°C
Wash Buffer (25X)	30ml	4°C
Plate Sealer	5	-

Other materials 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

Protein Information


Uniprot:	P49662
UniProt Protein Function:	CASP4: Involved in the activation cascade of caspases responsible for apoptosis execution. Cleaves caspase-1. Heterotetramer that consists of two anti-parallel arranged heterodimers, each one formed by a small and a large subunit. Widely expressed, with highest levels in spleen and lung. Moderate expression in heart and liver, low expression in skeletal muscle, kidney and testis. Not found in the brain. Belongs to the peptidase C14A family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Code:	P49662
NCBI GenInfo Identifier:	1352420
NCBI Gene ID:	837
NCBI Accession:	P49662.1
UniProt Related Accession:	P49662
Molecular Weight:	36,705 Da

Protocol

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

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Procedure
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.

Sample Type

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.

Caspase 4 Background

Caspase 4 is a member of the caspase family of proteins, which are enzymes involved in programmed cell death, also known as apoptosis. It is specifically classified as an inflammatory caspase due to its role in initiating inflammatory responses in certain cellular processes.

Function of Caspase 4

Caspase 4 is primarily associated with the innate immune response and plays a crucial role in host defense against bacterial infections. It is activated in response to certain bacterial components, specifically the lipopolysaccharides (LPS) found in the outer membrane of Gram-negative bacteria. Upon activation, caspase 4 initiates a cascade of cellular events that lead to the production and release of pro-inflammatory cytokines, such as interleukin-1 beta (IL-1β) and interleukin-18 (IL-18). These cytokines are essential for triggering immune responses and recruiting immune cells to combat the infection.

Cellular Localization and Activation of Caspase 4

Caspase 4 is primarily localized in the endoplasmic reticulum (ER), a cellular organelle involved in protein synthesis, folding, and quality control. Upon recognition of LPS, caspase 4 is activated and undergoes autoprocessing, resulting in its active form. This activation is facilitated by direct interaction between caspase 4 and LPS, leading to the recruitment and activation of downstream signaling molecules.

Caspase 4 in Inflammatory Diseases

In addition to its role in bacterial infections, caspase 4 has been implicated in various inflammatory diseases. Dysregulation of caspase 4 activation and function has been observed in conditions such as sepsis, inflammatory bowel disease, and neuroinflammatory disorders. In these diseases, caspase 4-mediated inflammatory responses can contribute to tissue damage and disease progression.

Caspase 4 FAQs

What is the Caspase 4 ELISA Kit?

The Human Caspase 4 ELISA Kit is a diagnostic tool that measures the levels of caspase 4, an enzyme involved in programmed cell death, in biological samples.

What are the advantages of using the Caspase 4 ELISA Kit?

The Human Caspase 4 ELISA Kit offers specificity, sensitivity, and quantitative results, allowing precise measurement of caspase 4 levels in various samples.

Where can I find more information about the Caspase 4 ELISA Kit?

For more detailed information about the Caspase 4 ELISA Kit, including technical specifications, performance characteristics, and ordering details, please refer to the product brochure or contact our customer support team. We are here to assist you with any inquiries you may have.

Illuminating the Multifaceted Role of Acetylation: Bridging Chemistry and Biology Introduction:

Understanding IgA Test: Importance, Procedure, and Interpretation

Biomarker Testing: Advancements, Applications, and Future Directions

Human Caspase 4 ELISA Kit

Description