Human Actinin Alpha 2 (ACTN2) ELISA Kit

two product lines: Traditional ELISA Kit and Ready-to-Use ELISA Kit.

	Traditional ELISA Kit	Ready-to-Use ELISA KIT
Product name:	Human Actinin Alpha 2 (ACTN2) ELISA Kit
Method:	Sandwich
Synonyms:	Alpha-actinin skeletal muscle isoform 2; F-actin cross-linking protein
Detection range:	0.312-20ng/mL
Target Protein:	ACTN2
Size:	96T/48T
Quality guarantee period:	for 12 months, 16 months
Catalog number:	DL-ACTN2-Hu (traditional)	(ready-to-use)
Assay length	1-4.5Hours	1-3.5Hours
Advantages:	Competitive price. High sensitivity. High stability. 12 months shelf life.	Pre-diluted Detection Reagent A and B Reduction in the number of steps when conducting the test All the reagents can be stored at -20℃ Faster reaction compare to other brands 16 months shelf life
Instruction Manual

1. Overview

Other names：Alpha-actinin skeletal muscle isoform 2; F-actin cross-linking protein

Function: F-actin cross-linking protein which is thought to anchor actin to a variety of intracellular structures. This is a bundling protein.

Sequence：
MNQIEPGVQY  NYVYDEDEYM  IQEEEWDRDL  LLDPAWEKQQ  RKTFTAWCNS
HLRKAGTQIE  NIEEDFRNGL  KLMLLLEVIS  GERLPKPDRG  KMRFHKIANV
NKALDYIASK  GVKLVSIGAE  EIVDGNVKMT  LGMIWTIILR  FAIQDISVEE
TSAKEGLLLW  CQRKTAPYRN  VNIQNFHTSW  KDGLGLCALI  HRHRPDLIDY
SKLNKDDPIG  NINLAMEIAE  KHLDIPKMLD  AEDIVNTPKP  DERAIMTYVS
CFYHAFAGAE  QAETAANRIC  KVLAVNQENE  RLMEEYERLA  SELLEWIRRT
IPWLENRTPE  KTMQAMQKKL  EDFRDYRRKH  KPPKVQEKCQ  LEINFNTLQT
KLRISNRPAF  MPSEGKMVSD  IAGAWQRLEQ  AEKGYEEWLL  NEIRRLERLE
HLAEKFRQKA  STHETWAYGK  EQILLQKDYE  SASLTEVRAL  LRKHEAFESD
LAAHQDRVEQ  IAAIAQELNE  LDYHDAVNVN  DRCQKICDQW  DRLGTLTQKR
REALERMEKL  LETIDQLHLE  FAKRAAPFNN  WMEGAMEDLQ  DMFIVHSIEE
IQSLITAHEQ  FKATLPEADG  ERQSIMAIQN  EVEKVIQSYN  IRISSSNPYS
TVTMDELRTK  WDKVKQLVPI  RDQSLQEELA  RQHANERLRR  QFAAQANAIG
PWIQNKMEEI  ARSSIQITGA  LEDQMNQLKQ  YEHNIINYKN  NIDKLEGDHQ
LIQEALVFDN  KHTNYTMEHI  RVGWELLLTT  IARTINEVET  QILTRDAKGI
TQEQMNEFRA  SFNHFDRRKN  GLMDHEDFRA  CLISMGYDLG  EAEFARIMTL
VDPNGQGTVT  FQSFIDFMTR  ETADTDTAEQ  VIASFRILAS  DKPYILAEEL
RRELPPDQAQ  YCIKRMPAYS  GPGSVPGALD  YAAFSSALYG  ESDL

2. Features

INTENDED USE
The kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of ACTN2 in human tissue homogenates and other biological fluids.

DETECTION RANGE
0.312-20ng/mL. The standard curve concentrations used for the ELISA’s were 20ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL, 0.312ng/mL.

SENSITIVITY
The minimum detectable dose of ACTN2 is typically less than 0.112ng/mL.
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. It was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.

SPECIFICITY
This assay has high sensitivity and excellent specificity for detection of ACTN2.
No significant cross-reactivity or interference between ACTN2 and analogues was observed.
Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between ACTN2 and all analogues, therefore, cross reactivity may still exist.

IMPORTANT NOTES
1. Limited by the current conditions and scientific technology, it is impossible to conduct comprehensive identification and analysis tests on the raw materials provided by suppliers. As a result, it is possible there are some qualitative and/or technical risks.
2. The final experimental results will be closely related to the validity of the products, operation skills of the end users and the experimental environments. Please make sure that sufficient samples are available to obtain accurate results.
3. Kits from different batches may be a little different in detection range, sensitivity and color developing time. Please perform the experiment exactly according to the instruction manual included in your kit. Electronic ones on our website are for reference only.
4. Do not mix or substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.
5. Protect all reagents from strong light during storage and incubation. All bottle caps of reagents should be closed tightly to prevent evaporation of liquids and contamination by microorganisms.
6. There may be a foggy substance in the wells when the plate is opened at the first time. It will not have any effect on the final assay results. Do not remove microtiter plate from the storage bag until needed.
7. Incorrect procedures during reagent preparation and loading, as well as incorrect parameter setting for the plate reader may lead to incorrect results. A microplate plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 O.D. or greater at 450 ± 10nm wavelength is acceptable for use in absorbance measurement. Please read the instruction carefully and adjust the instrument prior to the experiment.
8. Even the same experimenter may get different results from two separate experiments. In order to get better reproducible results, the operation of every step in the assay should be controlled. Furthermore, a preliminary experiment before the general assay for each batch is recommended.
9. Each kit has undergone several rigorous quality control tests. However, results from end users might be inconsistent with our in-house data due to some unexpected transportation conditions or different lab equipment. Intra-assay variance among kits from different batches could arise from the above factors as well.
10. Kits from different manufacturers with the same item might produce different results, since we have not compared our products with other manufacturers.
11. The standard in this kit, as well as the antigens used in antibody preparation are typically recombinant proteins. Differently expressed sequences, expression systems, and/or purification methods can be used in the preparation of recombinant proteins. There is also the possibility of differences in the screening technique of antibodies and antibody pairs in our kits. As a result, we cannot guarantee that our kit will be able to detect recombinant proteins produced by other companies. We do NOT recommend using our ELISA kits for the detection of other recombinant proteins.
12. Validity period: 12 months.
13. The instruction manual also works with the 48T kit, but all reagents in the 48T kit are reduced by half.

You can reference link of the kit as following
https://www.dldevelop.com/uploadfile/data/DL-ACTN2-Hu.pdf

Introduction

Item	Standard		Test
Description	The kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of ACTN2 in human tissue homogenates or other biological fluids.		Conform
Identification	Colorimetric		Positive
Composition	Traditional ELISA Kit	Ready-to-Use ELISA KIT	Conform
	Pre-coated, ready to use 96-well strip plate 1	Pre-coated, ready to use 96-well strip plate 1
	Plate sealer for 96 wells 2	Plate sealer for 96 wells 2
	Standard 2	Standard 2
	Diluents buffer 1×45mL	Standard Diluent 1×20mL
	Detection Reagent A 1×120μL	Detection Solution A 1×12mL
	Detection Reagent B 1×120μL	Detection Solution B 1×12mL
	TMB Substrate 1×9mL	TMB Substrate 1×9mL
	Stop Solution 1×6mL	Stop Solution 1×6mL
	Wash Buffer (30 × concentrate) 1×20mL	Wash Buffer (30 × concentrate) 1×20mL
	Instruction manual 1	Instruction manual 1

Test principle

The microtiter plate provided in this kit has been pre-coated with an antibody specific to the index. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to the index. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain the index, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of the index in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Recovery

Matrices listed below were spiked with certain level of recombinant ACTN2 and the recovery rates were calculated by comparing the measured value to the expected amount of the index in samples.

Matrix	Recovery range (%)	Average(%)
serum(n=5)	81-93	86
EDTA plasma(n=5)	80-97	88
heparin plasma(n=5)	90-101	95

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of the index and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1：2	1：4	1：8	1：16
serum(n=5)	82-96%	83-98%	81-99%	93-101%
EDTA plasma(n=5)	88-101%	86-95%	90-102%	80-93%
heparin plasma(n=5)	80-91%	82-90%	95-104%	79-95%

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level the index were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level the index were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

Stability

The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage conditions.
Note:
To minimize unnecessary influences on the performance, operation procedures and lab conditions, especially room temperature, air humidity and incubator temperatures should be strictly regulated. It is also strongly suggested that the whole assay is performed by the same experimenter from the beginning to the end.

Assay procedure summary

1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 2 hours at 37℃;
3. Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at 37℃;
4. Aspirate and wash 3 times;
5. Add 100µL prepared Detection Reagent B. Incubate 1 hour at 37℃;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 15-25 minutes at 37℃;
8. Add 50µL Stop Solution. Read at 450nm immediately.