Human GPBB/PYGB ELISA Kit (HUFI00822)- High Sensitivity

Description

Description

Human GPBB / PYGB ELISA Kit

GPBB, or PYGB, is a protein that controls the rate of glycogen breakdown and glucose release during exercise. This protein is found in the erythrocytes of vertebrates, which are red blood cells. It is also present in muscle tissue, where it helps regulate the flow of glucose from glycogen stores to muscles for fuel. It has been found to be a marker for different diseases such as diabetes, heart disease, Alzheimer's disease, and cancer. The Assay Genie Human GPBB/PYGB ELISA kit is a highly sensitive assay for the quantitative measurement of GPBB/PYGB in serum, 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 GPBB / PYGB ELISA Kit
Product Code:	HUFI00822
Size:	96 Assays
Alias:	PYGB, Glycogen phosphorylase brain form, Glycogen Phosphorylase BB, GPBB, Brain glycogen phosphorylase, Glycogen phosphorylase B, Glycogen Phosphorylase Isoenzyme BB, Phosphorylase glycogen brain
Detection Method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human PYGB concentrations in serum plasma and other biological fluids.
Sensitivity:	9.375pg/ml
Range:	15.625-1000pg/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 PYGB and the recovery rates were calculated by comparing the measured value to the expected amount of Human PYGB in samples.


Matrix	Recovery Range (%)	Average (%)
serum(n=5)	85-104	92
EDTA plasma(n=5)	88-103	96
UFH plasma(n=5)	92-98	96

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human PYGB 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)	88-105%	85-103%	85-105%
EDTA plasma(n=5)	82-94%	83-100%	92-97%
UFH plasma(n=5)	84-100%	90-99%	89-99%

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

Heading 1	Heading 2
UniProt	P11216
UniProt Protein Function	PYGB: Phosphorylase is an important allosteric enzyme in carbohydrate metabolism. Enzymes from different sources differ in their regulatory mechanisms and in their natural substrates. However, all known phosphorylases share catalytic and structural properties. Belongs to the glycogen phosphorylase family.
NCBI GenInfo Identifier	20178317
NCBI Gene ID	5834
Molecular Weight	96,696 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.

GPBB / PYGB Background

Glycogen

Glycogen is a complex form of sugar that animals use to store energy, just like plants store energy as starch. It is absent in plant tissues, with its highest concentration found in the liver, although skeletal muscles contain the largest glycogen stores by weight. Other tissues, including the kidney, heart, and brain, also contain glycogen, albeit in lower levels. Significantly, glycogen plays a pivotal role in maintaining glucose homeostasis in animals. Its metabolism is primarily regulated by insulin and glucagon, as well as molecules within their downstream signaling pathways. Insulin promotes glycogen synthesis, while glucagon stimulates its breakdown.

Glycogen Phosphorylase BB

Glycogen Phosphorylaase BB is an essential enzyme which is involved in the regulation of carbohydrate metabolism by mobilization of glycogen. Three different isoenzymes of glycogen phosphorylase (GP) exist; GPMM (present in muscles), GPLL (liver) and GPBB (brain and heart muscles). Glycogen phosphorylase-BB appears to be released into the circulation 2–4 h after myocardial injury. GPBB serves as an important tool in the field of cardiovascular research and clinical practice, contributing to a better understanding of cardiac pathologies and guiding appropriate treatment strategies. Its measurement, along with other cardiac biomarkers, helps in the timely intervention and management of heart-related disorders, ultimately leading to improved patient outcomes.

GPBB/PYGB ELISA Kit FAQs

What is the Human GPBB/PYGB ELISA kit used for?

The Human GPBB/PYGB ELISA kit is specifically designed for the quantitative measurement of Glycogen Phosphorylase BB (GPBB) and Glycogen Phosphorylase Brain (PYGB) levels in human samples. GPBB and PYGB are crucial enzymes involved in glycogen metabolism and have implications in various physiological and pathological processes. This ELISA kit enables researchers and clinicians to investigate the levels of GPBB and PYGB in biological samples, contributing to cardiovascular research, metabolic studies, and the understanding of glycogen-related disorders.

What are the advantages of using GPBB/PYGB ELISA Kit?

The GPBB/PYGB ELISA Kit offers several advantages for accurate and precise FSH measurements. It provides high sensitivity and specificity, ensuring reliable results. The kit is user-friendly, offering a streamlined protocol for easy handling and efficient analysis. Additionally, it provides a wide dynamic range, allowing for the detection of both low and high GPBB/PYGB levels, and it is suitable for both research and clinical applications.

What sample types are compatible with GPBB/PYGB ELISA kit?

The GPBB/PYGB ELISA Kit is compatible with various sample types, including serum, plasma, cell lysates, and tissue homogenates. It provides flexibility in sample selection, allowing researchers to analyze FSH levels in different biological matrices.

What are the storage requirements for the GPBB/PYGB ELISA Kit?

The GPBB/PYGB ELISA Kit components should be stored according to the instructions provided in the kit manual. Generally, it is recommended to store the kit components at the recommended temperature to ensure their stability and optimal performance.

What should I do if my assay results are not optimal?

If you encounter any issues or have suboptimal assay results, we recommend contacting our dedicated support team for assistance. They will be available to provide troubleshooting guidance, answer your questions, and ensure you achieve the best possible results with the GPBB/ PYGB ELISA Kit.

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Human GPBB / PYGB ELISA Kit

Description