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Rat IL-6 ELISA Kit (For Lysates)
Catalog Number: ELR-IL6-CL
User Manual
Last Revised: January 22, 2024
Protocol Video

Please read entire manual carefully before starting experiment.

Introduction
The RayBio® Rat IL-6 ELSA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked
immunosorbent assay for the quantitative measurement of Rat IL-6 cell lysate and tissue lysate. Thisassay employs an antibody specific for Rat IL-6 coated on a 96-well plate. Standards and samples are
pipetted into the wells and IL-6 present in a sample is bound to the wells by the immobilized antibody. Thewells are washed and biotinylated anti-Rat IL-6 antibody is added. After washing away unboundbiotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, aTMB substrate solution is added to the wells and color develops in proportion to the amount of IL-6 bound.
The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450nm.
Short on sample, or need higher sensitivity? Check out the IQELISA® Immuno-PCR assay platform
and our Simoa® Single Molecule Protein Detection Services .

Storage
The entire kit may be stored at -20°C for up to 1 year from the date of shipment. Avoid repeated freezethaw cycles. The kit may be stored at 4°C for up to 6 months. For extended storage, it is recommended tostore at -80°C. For prepared reagent storage, see table below.

For Research Use Only1Reagents
Component

Size / Description

Storage / Stability
After Preparation

Rat IL-6 Microplate

96 wells (12 strips x 8 wells) coated with anti-RatIL-6.

1 month at 4°C*

Rat IL-6 Standard Protein

2 vials of Rat IL-6. 1 vial is enough to run eachstandard in duplicate.

1 week at -80°C

Rat IL-6 Detection Antibody

2 vials of biotinylated anti-Rat IL-6. Each vial isenough to assay half the microplate.

5 days at 4°C

Wash Buffer

25 ml of 20X concentrated solution.

1 month at 4°C

HRP-Streptavidin

200 µl 400X concentrated HRP-conjugatedstreptavidin.

Do not store andreuse.

TMB One-Step SubstrateReagent

12 ml of 3,3,5,5'-tetramethylbenzidine (TMB) inbuffer solution.

N/A

Stop Solution

8 ml of 0.2 M sulfuric acid.

N/A

Sample Diluent Buffer

10 ml of 5X concentrated buffer.

1 month at 4°C

Assay Diluent B

15 ml of 5X concentrated buffer.

1 month at 4°C

Lysis Buffer

5 ml of 2X cell lysate buffer.

1 month at 4°C*Return unused wells to the pouch containing desiccant pack, reseal along entire edge.

Additional Materials Required
1. Microplate reader capable of measuring absorbance at 450 nm.
2. Precision pipettes to deliver 2 µl to 1 ml volumes.
3. Adjustable 1-25 ml pipettes for reagent preparation.
4. 100 ml and 1 liter graduated cylinders.
5. Absorbent paper.
6. Distilled or deionized water.
7. Log-log graph paper or computer and software for ELISA data analysis.
8. Tubes to prepare standard or sample dilutions.

For Research Use Only2

Reagent Preparation
1. Bring all reagents and samples to room temperature (18 - 25°C) before use.
2. Sample Diluent Buffer and Assay Diluent B should be diluted 5-fold with deionized or distilled water
before use. Lysis Buffer should be diluted 2-fold with deionized or distilled water (for cell lysate andtissue lysate).
3. Sample dilution: Tissue lysate and cell lysate samples should be diluted at least 5-fold with 1X
Sample Diluent Buffer. Generally we recommend a minimum of 1 mg of protein per 1 ml of original
lysate solution, though more concentrated is better. We also recommend the addition of proteaseinhibitors (not included) to the lysis buffer prior to use. Detailed recommendations on lysis
preparation may be found here: www.raybiotech.com/tips-on-sample-preparation.html
Note: Levels of IL-6 may vary between different samples. Optimal dilution factors for each sample
must be determined by the investigator.
4. Preparation of standard: Briefly spin a vial of standard protein. Add 500 µl 1X Sample Diluent Buffer
into the Standard Protein vial to prepare a 10,000 pg/ml standard solution. Dissolve the powder
thoroughly by a gentle mix. Pipette 300 µl 1X Sample Diluent Buffer into each tube. Use the 10,000pg/ml standard solution to produce a dilution series (shown below). Mix each tube thoroughly before
the next transfer. 1X Sample Diluent Buffer serves as the zero standard (0 pg/ml).
200 µl200 µl200 µl200 µl200 µl200 µl

Std1

Std2

Std3

Std4

Std5

Std6

Std7

ZeroStandard

Diluent
volumeStandard +500 µl

300 µl300 µl300 µl300 µl300 µl300 µl300 µl

Conc.

10000
pg/ml

4000
pg/ml

1600
pg/ml

102.4
pg/ml

40.96
pg/ml

0pg/ml640 pg/ml 256 pg/ml

5. If the Wash Buffer (20X) contains visible crystals, warm to room temperature and mix gently until
dissolved. Dilute 20 ml of Wash Buffer into deionized or distilled water to yield 400 ml of 1X WashBuffer.
6. Briefly spin the Detection Antibody vial before use. Add 100 µl of 1X Assay Diluent B into the vial toprepare a detection antibody concentrate. Pipette up and down to mix gently (the concentrate can be
stored at 4°C for 5 days). The detection antibody concentrate should be diluted 80-fold with 1X
Assay Diluent B and used in step 5 of Part VI Assay Procedure.

For Research Use Only3

7. Briefly spin the HRP-Streptavidin concentrate vial and pipette up and down to mix gently before use,
as precipitates may form during storage. HRP-Streptavidin concentrate should be diluted 400-foldwith 1X Assay Diluent B.
For example: Briefly spin the HRP-Streptavidin vial and pipette up and down to mix gently. Add 30 µlof HRP-Streptavidin concentrate into a tube with 12 ml 1X Assay Diluent B to prepare a 400-folddiluted HRP- Streptavidin solution (don't store the diluted solution for next day use). Mix well.

Assay Procedure
1. Bring all reagents and samples to room temperature (18 - 25°C) before use. It is recommended thatall standards and samples be run at least in duplicate.
2. Label removable 8-well strips as appropriate for your experiment.
3. Add 100 µl of each standard (see Reagent Preparation step 4) and sample into appropriate wells.
Cover wells and incubate for 2.5 hours at room temperature with gentle shaking.
4. Discard the solution and wash 4 times with 1X Wash Solution. Wash by filling each well with Wash
Buffer (300 µl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each stepis essential to good performance. After the last wash, remove any remaining Wash Buffer byaspirating or decanting. Invert the plate and blot it against clean paper towels.
5. Add 100 µl of 1X prepared biotinylated antibody (Reagent Preparation step 6) to each well. Incubate
for 1 hour at room temperature with gentle shaking.
6. Discard the solution. Repeat the wash as in step 4.
7. Add 100 µl of prepared Streptavidin solution (see Reagent Preparation step 7) to each well. Incubate
for 45 minutes at room temperature with gentle shaking.
8. Discard the solution. Repeat the wash as in step 4.
9. Add 100 µl of TMB One-Step Substrate Reagent to each well. Incubate for 30 minutes at room
temperature in the dark with gentle shaking.
10. Add 50 µl of Stop Solution to each well. Read at 450 nm immediately.

Assay Procedure Summary
1. Prepare all reagents, samples and standards as instructed.
2. Add 100 µl standard or sample to each well. Incubate 2.5 hours at room temperature.
3. Add 100 µl prepared biotin antibody to each well. Incubate 1 hour at room temperature.
4. Add 100 µl prepared Streptavidin solution. Incubate 45 minutes at room temperature.
5. Add 100 µl TMB One-Step Substrate Reagent to each well. Incubate 30 minutes at roomtemperature.
6. Add 50 µl Stop Solution to each well. Read at 450 nm immediately.

For Research Use Only4

Calculation of Results
Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract
the average zero standard optical density. Plot the standard curve on log-log graph paper or using Sigma
plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit
straight line through the standard points.
A. Typical Data
These standard curves are for demonstration only. A standard curve must be run with each assay.

B. Sensitivity
The minimum detectable dose of Rat IL-6 was determined to be 15 pg/ml.
Minimum detectable dose is defined as the analyte concentration resulting in an absorbance that is 2
standard deviations higher than that of the blank (diluent buffer).
C. Spiking & Recovery
Recovery was determined by spiking various levels of Rat IL-6 into the sample types listed below. Mean
recoveries are as follows:

For Research Use Only

5D. Linearity

E. Reproducibility
Intra-Assay CV%: <10%Inter-Assay CV%: <12%Specificity
The antibody pair provided in this kit recognizes rat IL-6.

For Research Use Only

6

Troubleshooting Guide
Problem

Cause

Solution

Inaccurate pipetting
Improper standard dilution

Check pipettes
Briefly centrifuge the standard
protein and dissolve the powder
thoroughly by gently mixing

Low signal

Improper preparation of standardand/or biotinylated antibody
Too brief incubation times
Inadequate reagent volumes orimproper dilution

Briefly spin down vials before
opening. Dissolve the powder
thoroughly.
Ensure sufficient incubation time.
Assay procedure step 3 may bedone overnight at 4°C with gentle
shaking (note: may increase
overall signals includingbackground).
Check pipettes and ensure
correct preparation

Large CV

Inaccurate pipetting
Air bubbles in wells

Check pipettes
Remove bubbles in wells

Plate is insufficiently washedContaminated wash buffer

Review the manual for proper
wash. If using a plate washer,ensure that all ports areunobstructed.
Make fresh wash buffer

Improper storage of the ELISA kit
Stop solution

Store your standard at <-70°C
after reconstitution, others at 4°C.
Keep substrate solution protectedfrom light.
Add stop solution to each wellbefore reading plate

Poor standard curveHigh backgroundLow sensitivity

For Research Use Only7