Human XBP1 ELISA Kit (HUFI02090)- High Sensitivity

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 XBP1 ELISA Kit
Product Code:	HUFI02090
Size:	96 Assays
Alias:	XBP1, HTF, TREB5, XBP2, TREB5, X-box binding protein 1, X-box-binding protein 1, XBP-1, XBP2Tax-responsive element-binding protein 5
Detection Method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human XBP1 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 XBP1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human XBP1 in samples.


Matrix	Recovery Range (%)	Average (%)
Serum (n=5)	87-105	96
EDTA Plasma (n=5)	87-102	93
UFH Plasma (n=5)	88-103	95

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human XBP1 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)	85-102%	88-93%	91-100%
EDTA Plasma (n=5)	86-98%	82-95%	83-100%
UFH Plasma (n=5)	83-93%	84-98%	83-94%

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

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 Preparation

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.

XBP1 Background

X Box Binding Protein-1 (XBP1)

XBP1 (X-box binding protein 1) is a protein from the bZIP domain containing protein family, that plays a crucial role in the regulation of the unfolded protein response (UPR).

The unfolded protein response (UPR) is a cellular mechanism that is activated in response to stress within the endoplasmic reticulum (ER). When the ER encounters an accumulation of unfolded or misfolded proteins, it triggers the UPR to restore ER homeostasis. The UPR achieves this by increasing the production of chaperone proteins that assist in protein folding, reducing protein synthesis, and promoting the degradation of misfolded proteins. If the ER stress cannot be resolved, the UPR can also induce cell death to eliminate damaged cells.

It is also required for embryonic cardiac myogenesis and hepatogenesis, along with the development of secretory tissues such as exocrine pancreas and salivary gland.

XBP1 Gene

The gene that encodes XBP1 is also known as XBP1 or XBP1s (spliced form). Located in chromosome 22 (position 22q12), this gene codes for a trasncription actor that regulates MHC class II genes by binding to a promoter element referred to as an X box. The product of this gene is a bZIP (the basci leucine zipper doman containing) protein, which has been identified as a transciption factor that binds to and enhaces T cell leukemia virus type 1 promoter. It is also thought to increase viral protein expression by acting as the DNA binding partner of a viral transactivator.

XBP1 Protein

XBP1 is a transcription factor, meaning it is involved in the regulation of gene expression. It is activated in response to ER stress, which occurs when the folding of proteins in the ER is disrupted or overwhelmed. When activated, XBP1 moves to the nucleus of the cell and binds to specific DNA sequences known as the X-box motif, thereby activating the transcription of genes involved in various aspects of the UPR.

The downstream targets of XBP1 include chaperones, enzymes involved in protein folding and ER-associated degradation, as well as components of the ER-associated protein degradation (ERAD) machinery. By activating these target genes, XBP1 helps restore ER homeostasis and promote cell survival under conditions of ER stress.

XBP1 (X box binding protein-1 ) Structure. Source: Uniprot

XBP1 splicing and activation

One of the notable features of XBP1 is its unique activation mechanism. Initially, XBP1 is synthesized as an inactive precursor called XBP1u (unspliced form) in the cytoplasm. However, during ER stress, an ER transmembrane protein called IRE1 (inositol-requiring enzyme 1), which possesses endoribonuclease activity, is activated. IRE1 cleaves XBP1u mRNA at a specific site, resulting in the removal of an intron. This unconventional XBP1 splicing event generates the active form of XBP1, known as XBP1s. XBP1s is then translated into a functional protein that can regulate the expression of target genes involved in protein folding, ER-associated degradation, and other UPR-related processes. This mechanism ensures that XBP1 is specifically activated in response to ER stress, contributing to the cellular adaptation to maintain protein folding integrity.

XBP1 Fact Sheet


Gene Name	XBP1
Gene ID	7494
Uniprot ID	P17861
Full Name	X-box-binding protein 1
Protein Family	bZIP Family
Molecular Weight	54 kDa

Human XBP1 ELISA kit FAQs

Q: What does the Human XBP1 ELISA kit measure?

The Human XBP1 ELISA kit measures the levels of XBP1 protein in human samples such as serum, plasma, cell lysates, and other biological fluids. It provides quantitative data on XBP1 concentration, allowing researchers to assess its expression in various samples.

Q: Can this kit be used for other species than human??

This particular ELISA kit is specifically designed and validated for the detection of human XBP1 protein. For other species, it is recommended to use ELISA kits specifically developed and validated for those species.

Q: Where can I find additional technical support or assistance with the Human XBP1 ELISA kit?

For any technical inquiries or assistance regarding the UNC13A ELISA kit, you can reach out to our team. They will be available to answer your questions and provide the necessary guidance to ensure a successful experiment.

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