EH8788

Human GJB6(Gap junction beta-6 protein) ELISA Kit
Catalogue No.: EH8788

Revision: V 3.0
Size: 48T/96T
Reactivity: Human
Range: 15.625-1000pg/ml
Sensitivity: 9.375pg/ml
Application: For quantitative detection of GJB6 in cell lysates, tissue homogenates and other biological fluids.
Storage: 2-8°C
Expiry Date: see kit label
Principle: Sandwich
NOTE: FOR RESEARCH USE ONLY.
Kit Components
No. Item Specifications(48T/96T) Storage

E001 ELISA Microplate(Dismountable) 8×6/8×12 2-8°C/-20°C
E002 Lyophilized Standard 1vial/2vial 2-8°C/-20°C
E039 Sample Dilution Buffer 10ml/20ml 2-8°C
E003 Biotin-labeled Antibody(Concentrated) 60ul/120ul 2-8°C(Avoid Direct Light)
E040 Antibody Dilution Buffer 5ml/10ml 2-8°C
E034 HRP-Streptavidin Conjugate(SABC) 60ul/120ul 2-8°C(Avoid Direct Light)
E049 SABC Dilution Buffer 5ml/10ml 2-8°C
E024 TMB Substrate 5ml/10ml 2-8°C(Avoid Direct Light)
E026 Stop Solution 5ml/10ml 2-8°C
E038 Wash Buffer(25X) 15ml/30ml 2-8°C
E006 Plate Sealer 3/5pieces
E007 Product Description 1copy
1
Wuhan Fine Biotech Co., Ltd.
1st Floor, Building A02, Optics Valley Biomedical Industrial Park, Wuhan, China (430075)
Tel: (0086)027-87384275 Fax: (0086)027-87800889 www.fn-test.com
Typical Data & Standard Curve
Results of a typical standard operation of a GJB6 ELISA Kit are listed below. This standard curve was generated at
our lab for demonstration purpose only. Users shall obtain standard curve as per experiment by themselves.
(N/A=not applicable)
STD.(pg/ml) OD-1 OD-2 Average Corrected
0 0.123 0.127 0.125 0
15.625 0.302 0.31 0.306 0.181
31.25 0.517 0.533 0.525 0.4
62.5 0.703 0.723 0.713 0.588
125 1.276 1.314 1.295 1.17
250 1.526 1.57 1.548 1.423
500 2.091 2.151 2.121 1.996
1000 2.376 2.444 2.41 2.285
2
Specificity
This assay has high sensitivity and excellent specificity for detection of GJB6. No significant cross-reactivity or
interference between GJB6 and analogues was observed.
Note: Limited by current skills and knowledge, it is difficult for us to complete the cross-reactivity detection
between GJB6 and all the analogues, therefore, cross reaction may still exist.
Recovery
Matrices listed below were spiked with certain level of GJB6 and the recovery rates were calculated by comparing
the measured value to the expected amount of GJB6 in samples.
Matrix Recovery Range (%) Average (%)
cell lysates (n=5) 90-105 95
Precision
Intra-Assay: CV<8%
Inter-Assay: CV<10%
Stability
The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 10% within
the expiration date under appropriate storage condition.
Standard(n=5) 37°C for 1 month 2-8°C for 6 months
Average (%) 80 95-100
To minimize extra influence on performance, operation procedures and lab conditions, especially room
temperature, air humidity, incubator temperature should be strictly controlled. It is strongly suggested that the
same operator performs the whole assay from the beginning to the end.
3
Operation Procedure
Elisa Kits Operation Guide & Targeted control of TMB coloring
https://www.fn-test.com/videos/elisa-test/ & www.fn-test.com/category/knowledge-share/
Principle of the Assay

This kit was based on sandwich enzyme-linked immune-sorbent assay technology. Capture antibody was pre-coated onto 96-well
plates. And the biotin conjugated antibody was used as detection antibodies. The standards, test samples and biotin conjugated
detection antibody were added to the wells subsequently, and washed with wash buffer. HRP-Streptavidin was added and unbound
conjugates were washed away with wash buffer. TMB substrates were used to visualize HRP enzymatic reaction. TMB was catalyzed
by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow is
proportional to the target amount of sample captured in plate. Read the O.D. absorbance at 450nm in a microplate reader, and then
the concentration of target can be calculated.
Precautions
1. To inspect the validity of experiment operation and the appropriateness of sample dilution proportion, pilot experiment using
standards and a small number of samples is recommended.
2. After opening and before using, keep plate dry.
3. Before using the kit, spin tubes and bring down all components to the bottom of tubes.
4. Storage TMB reagents avoid light.
5. Washing process is very important, not fully wash easily cause a false positive and high background.
6. Duplicate well assay is recommended for both standard and sample testing.
7. Don’t let microplate dry at the assay, for dry plate will inactivate active components on plate.
8. Don’t reuse tips and tubes to avoid cross contamination.
9. Please do not mix the reagents in different kits of our company. Do not mix reagents from other manufacturers.
10. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.
Material Required but Not Supplied

1. Microplate reader (wavelength:450nm)
2. 37°C incubator
3. Automated plate washer
4. Precision single and multi-channel pipette and disposable tips
5. Clean tubes and Eppendorf tubes
6. Deionized or distilled water
4
Washing
Manual: Discard the solution in the plate without touching the side walls. Clap the plate on absorbent filter papers or other
absorbent material. Fill each well completely with 350ul wash buffer and soak for 1 to 2 minutes, then aspirate contents from the
plate, and clap the plate on absorbent filter papers or other absorbent material.
Automatic: Aspirate all wells, and then wash plate with 350ul wash buffer. After the final wash, invert plate, and clap the plate on
absorbent filter papers or other absorbent material. It is recommended that the washer shall be set for soaking 1 minute. (Note: set
the height of the needles; be sure the fluid can be sipped up completely)
Sample Collection and Storage (universal)

https://www.fn-test.com/download/
Please refer to the following link or qr code for detailed sample preparation guidelines
● Serum: Place whole blood sample at room temperature for 2 hours or put it at 2-8°C overnight and centrifugation for 20
minutes at approximately 1000×g, Collect the supernatant and carry out the assay immediately. Blood collection tubes should
be disposable, non-pyrogenic, and non-endotoxin.
● Plasma: Collect plasma using EDTA-Na2 or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2-8°C
within 30 minutes of collection. Collect the supernatant and carry out the assay immediately. Avoid hemolysis, high cholesterol
samples.
● Tissue Homogenates: As hemolysis blood has relation to assay result, it is necessary to remove residual blood by washing
tissue with pre-cooling PBS buffer (0.01M, pH=7.4). Mince tissue after weighing it and get it homogenized in PBS (the volume
depends on the weight of the tissue. Normal, 9mL PBS would be appropriate to 1 gram tissue pieces. Some protease inhibitors
are recommended to add into the PBS) with a glass homogenizer on ice. To further break the cells, you can sonicate the
suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then centrifuged for 5
minutes at 5000×g to get the supernatant. The total protein concentration was determined by BCA kit and the total protein
concentration of each pore sample should not exceed 0.3mg.
● Adherent and Suspension Cell Culture: Use three T25 flasks or one T75 flask for cell culture, the number of cells (1x107);
1. Suspension cell: centrifuge at 2500 rpm at 2-8℃ for 5 minutes; collect clarified cell culture supernatant;
2. Adherent cell: collect supernatant directly; centrifuge at 2500 rpm at 2-8℃ for 5 minutes; collect clarified cell culture
supernatant for immediate detection or store it separately at -80℃.
● Cell Lysate Preparation: Two types of cell lysates are specified below.
1. Suspension Cell Lysate：Centrifuge at 2500 rpm at 2-8℃ for 5 minutes; Then add pre-cooling PBS into collected cell and
gently mix. Recollect cell by repeating centrifugation. Add 0.5-1ml RIPA lysis buffer (NP-40 lysis buffer or Triton X-100 surfactant
is not recommended due to the interfering with antigen-antibody reaction). Add suitable protease inhibitor (e.g. PMSF,
working concentration: 1mmol/L). Lyse the cell on ice for 30min-1h. During lysate process, use the tip for pipetting or
intermittently shake the centrifugal tube to completely lyse the protein. Alternatively, cells are subject to fragmentation by
ultrasonic cell disruptor (300W, 3~5 s/time, 30s intervals, four-five times) or ultrasonic generator (14μm for 30s ). At the end of
lysate or ultrasonic disruption, centrifuge at 10000rpm at 2-8℃ for 10 minutes. Then, the supernatant is added into EP tube
and stored at -80℃.
5
2. Adherent Cell Lysate: Absorb supernatant and add pre-cooling PBS once. Then, add 0.5-1ml RIPA lysis buffer (NP-40 lysis
buffer or Triton X-100 surfactant is not recommended due to the interfering with antigen-antibody reaction). Add the suitable
protease inhibitor (e.g. PMSF, working concentration: 1mmol/L). Scrape adherent cell gently with a cell scraper. Add the cell
suspension into centrifugal tube. Lyse the cell on ice for 30min-1h. During lysate process, use the tip for pipetting or
intermittently shake the centrifugal tube to completely lyse the protein. Alternatively, cells are subject to fragmentation by
ultrasonic generator (14μm for 30s ) or ultrasonic cell disruptor (300W, 3~5 s/time, 30s intervals, four-five times). At the end of
lysate/ultrasonic disruption, centrifuge at 10000rpm at 2-8℃ for 10 minutes. Then, the supernatant is added into EP tube and
stored at -80℃.
● Other Biological Fluids: Centrifuge samples for 20 minutes at 1000×g at 2-8°C. Collect supernatant and carry out the assay
immediately.
Note: Samples used within 5 days can be stored at 2-8°C; otherwise, they must be stored at -20°C or -80°C or liquid nitrogen to avoid
loss of biological activity and contamination. Avoid multiple freeze-thaw cycles. Hemolytic samples are not suitable for this test.
Sample Dilution
The user should estimate the concentration of target protein in the test sample, and select a proper dilution factor to make the
diluted target protein concentration fall in the optimal detection range of the kit. Dilute the sample with the provided dilution buffer,
and several trials may be necessary. The test sample must be well mixed with the dilution buffer. And also standard curves and
sample should be making in pre-experiment. If samples with very high concentrations, dilute samples with PBS first and then dilute
the samples with Sample Dilution.
The matrix components in the sample will affect the test results, which it need to be diluted at least 1/2 with Sample Dilution
Buffer before testing!
Reagent Preparation and Storage

Bring all reagents and samples to room temperature for 20 minutes before use.
1, Wash Buffer:
If crystals have formed in the concentrate, you can warm it with 40°C water bath (Heating temperature should not exceed 50°C) and
mix it gently until the crystals have completely been dissolved. The solution should be cooled to room temperature before use.
Dilute 30ml (15ml for 48T) Concentrated Wash Buffer to 750ml (375ml for 48T) Wash Buffer with deionized or distilled water
(The recommended resistivity of deionized or distilled water is 18MΩ). Put unused solution back at 2-8°C.
2, Standards:
1). Add 1 ml Sample Dilution Buffer into one Standard tube (labeled as zero tube), keep the tube at room temperature for 10 minutes
and mix them thoroughly.
Note: If the standard tube concentration higher than the range of the kit，please dilute it and labeled as zero tube.
2). Label 7 EP tubes with 1/2, 1/4, 1/8, 1/16, 1/32, 1/64 and blank respectively. Add 0.3ml of the Sample Dilution Buffer into each
tube. Add 0.3ml of the above Standard solution (from zero tube) into 1st tube and mix them thoroughly. Transfer 0.3ml from 1st tube
to 2nd tube and mix them thoroughly. Transfer 0.3ml from 2nd tube to 3rd tube and mix them thoroughly, and so on. Sample Dilution
Buffer was used for the blank control.
Note: It is best to use Standard Solutions within 2 hours.
6
3, Preparation of Biotin-labeled Antibody Working Solution:
Prepare it within 1 hour before experiment.
1）Calculate required total volume of the working solution: 0.1ml/well × quantity of wells. (Allow 0.1-0.2ml more than the total
volume.)
2）Dilute the Biotin-detection antibody with Antibody Dilution Buffer at 1:100 and mix them thoroughly. (i.e. Add 1ul Biotin-labeled
antibody into 99ul Antibody Dilution Buffer.)
4, Preparation of HRP-Streptavidin Conjugate (SABC) Working Solution:
Prepare it within 30 minutes before experiment.
1）Calculate required total volume of the working solution: 0.1ml/well × quantity of wells. (Allow 0.1-0.2ml more than the total
volume.)
2）Dilute the SABC with SABC Dilution Buffer at 1:100 and mix them thoroughly. (i.e. Add 1ul of SABC into 99ul of SABC Dilution
Buffer.)
Assay Procedure
When diluting samples and reagents, they must be mixed completely and evenly. Before adding TMB into wells, equilibrate TMB
Substrate for 30 minutes at 37°C. It is recommended to plot a standard curve for each test.
1. Set standard, test samples (diluted at least 1/2 with Sample Dilution Buffer), control (blank) wells on the pre-coated plate
respectively, and then, records their positions. It is recommended to measure each standard and sample in duplicate.
2. Prepare Standards: Aliquot 100ul of zero tube, 1sttube, 2ndtube, 3rdtube, 4thtube, 5thtube, 6thtube and Sample Dilution Buffer
(blank) into the standard wells.
3. Add Samples: Add 100ul of properly diluted sample into test sample wells.
4. Incubate: Seal the plate with a cover and incubate at 37°C for 90 minutes.
5. Wash: Remove the cover and discard the plate content, and wash plate 2 times with Wash Buffer. Do NOT let the wells dry
completely at any time.
6. Biotin-labeled Antibody: Add 100ul Biotin-labeled antibody working solution into above wells (standard, test sample and blank
wells). Add the solution at the bottom of each well without touching the sidewall, cover the plate and incubate at 37°C for 60
minutes.
7. Wash: Remove the cover, and wash plate 3 times with Wash Buffer, and let the Wash Buffer stay in the wells for 1-2 minutes
each time.
8. HRP-Streptavidin Conjugate (SABC): Add 100ul of SABC Working Solution into each well, cover the plate and incubate at 37°C
for 30 minutes.
9. Wash: Remove the cover and wash plate 5 times with Wash Buffer, and let the wash buffer stay in the wells for 1-2 minutes
each time.
10. TMB Substrate: Add 90ul TMB Substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 minutes.
(Note: The reaction time can be shortened or extended according to the actual color change, but not more than 30 minutes.
You can terminate the reaction when apparent gradient appeared in standard wells.)
11. Stop: Add 50ul Stop Solution into each well. The color will turn yellow immediately. The adding order of Stop Solution should
be as the same as the TMB Substrate Solution.
12. OD Measurement: Read the O.D. absorbance at 450nm in Microplate Reader immediately after adding the stop solution.
Regarding calculation, (the relative O.D.450) = (the O.D.450 of each well) – (the O.D.450 of blank well). The standard curve can be
plotted as the relative O.D.450 of each standard solution (Y) vs. the respective concentration of the standard solution (X). The target
concentration of the samples can be interpolated from the standard curve. It is recommended to use some professional software to
do this calculation, such as Curve Expert 1.3 or 1.4.
7
Note: If the samples measured were diluted, multiply the dilution factor to the concentrations from interpolation to obtain the
concentration before dilution.
Summary
Step1: Add 100ul standard or sample to each well and incubate for 90 minutes at 37°C.
Wash step: Aspirate and wash plates 2 times.
Step2: Add 100ul Biotin-labeled antibody working solution to each well and incubate for 60 minutes at 37°C.
Step3: Add 100ul SABC Working Solution into each well and incubate for 30 minutes at 37°C.
Step4: Add 90ul TMB Substrate Solution. Incubate 10-20 minutes at 37°C.
Step5: Add 50ul Stop Solution. Read at 450nm immediately and calculation.
8

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Human GJB6(Gap junction beta-6 protein) ELISA Kit

Catalogue No.: EH8788

No. Item Specifications(48T/96T) Storage

STD.(pg/ml) OD-1 OD-2 Average Corrected

0 0.123 0.127 0.125 0

15.625 0.302 0.31 0.306 0.181

31.25 0.517 0.533 0.525 0.4

62.5 0.703 0.723 0.713 0.588

125 1.276 1.314 1.295 1.17

250 1.526 1.57 1.548 1.423

500 2.091 2.151 2.121 1.996

1000 2.376 2.444 2.41 2.285

Matrix Recovery Range (%) Average (%)

cell lysates (n=5) 90-105 95

Standard(n=5) 37°C for 1 month 2-8°C for 6 months

Average (%) 80 95-100

Principle of the Assay

Material Required but Not Supplied

Sample Collection and Storage (universal)

Reagent Preparation and Storage

Note: It is best to use Standard Solutions within 2 hours.

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