Rat IL-6 ELISA kits allow researchers to feasibly study interleukin 6 in vivo
in animal models. IL-6 plays an important role in the immune response and
represents an important biomarker for immune system activity, which has
implications in the development of cancer, autoimmune disease and sepsis. IL-6
concentration may be elevated in patients suffering from immune diseases,
bacterial infections and viral infections. IL-6 concentration can also be
increased in tumors and thus IL-6 ELISA kits are used to detect IL-6 levels in
various types of cancers including breast cancer, endometrial cancer, ovarian
cancer, lung cancer and prostate cancer.
Cytokine with a wide variety of biological functions. It is a potent
inducer of the acute phase response. Plays an essential role in the
final differentiation of B-cells into Ig-secreting cells Involved in
lymphocyte and monocyte differentiation. Acts on B-cells, T-cells,
hepatocytes, hematopoietic progenitor cells and cells of the CNS.
Required for the generation of T(H)17 cells. Also acts as a myokine.
It is discharged into the bloodstream after muscle contraction and
acts to increase the breakdown of fats and to improve insulin
resistance. It induces myeloma and plasmacytoma growth and induces
nerve cells differentiation.
Molecular Weight:
24,357 Da
Component
Quantity (96 Assays)
Storage
ELISA Microplate (Dismountable)
8×12 strips
-20°C
Lyophilized Standard
2
-20°C
Sample Diluent
20ml
-20°C
Assay Diluent A
10mL
-20°C
Assay Diluent B
10mL
-20°C
Detection Reagent A
120µL
-20°C
Detection Reagent B
120µL
-20°C
Wash Buffer
30mL
4°C
Substrate
10mL
4°C
Stop Solution
10mL
4°C
Plate Sealer
5
-
Other materials and equipment 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
*Note: The below protocol is a sample protocol.
Protocols are specific to each batch/lot. For the correct instructions
please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the
reagents at 37°C directly). All the reagents should be mixed
thoroughly by gently swirling before pipetting. Avoid foaming. Keep
appropriate numbers of strips for 1 experiment and remove extra strips
from microtiter plate. Removed strips should be resealed and stored at
-20°C until the kits expiry date. Prepare all reagents, working
standards and samples as directed in the previous sections. Please predict
the concentration before assaying. If values for these are not within the
range of the standard curve, users must determine the optimal sample
dilutions for their experiments. We recommend running all samples in
duplicate.
Step
1.
Add Sample: Add 100µL of Standard, Blank, or Sample per well.
The blank well is added with Sample diluent. Solutions are added to
the bottom of micro ELISA plate well, avoid inside wall touching and
foaming as possible. Mix it gently. Cover the plate with sealer we
provided. Incubate for 120 minutes at 37°C.
2.
Remove the liquid from each well, don't wash. Add 100µL of
Detection Reagent A working solution to each well. Cover with the
Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate
for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy
warm to room temperature until solution is uniform.
3.
Aspirate each well and wash, repeating the process three times. Wash
by filling each well with Wash Buffer (approximately 400µL) (a
squirt bottle, multi-channel pipette,manifold dispenser or automated
washer are needed). Complete removal of liquid at each step is
essential. After the last wash, completely remove remaining Wash
Buffer by aspirating or decanting. Invert the plate and pat it against
thick clean absorbent paper.
4.
Add 100µL of Detection Reagent B working solution to each well.
Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.
Repeat the wash process for five times as conducted in step 3.
6.
Add 90µL of Substrate Solution to each well. Cover with a new
Plate sealer and incubate for 10-20 minutes at 37°C. Protect the
plate from light. The reaction time can be shortened or extended
according to the actual color change, but this should not exceed more
than 30 minutes. When apparent gradient appears in standard wells,
user should terminatethe reaction.
7.
Add 50µL of Stop Solution to each well. If color change does not
appear uniform, gently tap the plate to ensure thorough mixing.
8.
Determine the optical density (OD value) of each well at once, using a
micro-plate reader set to 450 nm. User should open the micro-plate
reader in advance, preheat the instrument, and set the testing
parameters.
9.
After experiment, store all reagents according to the specified
storage temperature respectively until their expiry.
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.
Alrashdi et al.
Effects of Rosmarinus officinalis L. Extract on Neurobehavioral and Neurobiological Changes in Male Rats with Pentylenetetrazol-Induced Epilepsy