User Manual - LSBio

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LSBioTM Human NOS2 / iNOSCell-Based ELISA KitCatalog No. LS-F2081User ManualPlease Read the Manual CarefullyBefore Starting your ExperimentFor research use only. Not approved for use in humans or for clinical diagnosis.

CONTENTSPAGEIntroduction.3Colorimetric Cell-Based ELISAsINOS Cell-Based ELISAAssay Format . . 4Assay Restrictions . 5Antibody Specificity.6Anti-INOS AntibodyAnti-GAPDH AntibodyMaterials Included.8Storage and Stability.8Buffer Preparation and Recommendations.9Additional Materials Required.11Health and Safety Precautions.11Experiment Design.12Assay Protocol .13Short Protocol 16Data Normalization .17

INTRODUCTIONColorimetric Cell-Based ELISAsThe Colorimetric Cell-Based ELISA Kit allows for the detection of varioustarget proteins and the effects that certain stimulation conditions have ontarget protein expression in different cell lines. Qualitative determination oftarget protein concentration is achieved by an indirect ELISA format. Inessence, the target protein is captured by target-specific primary (1 )antibodies while the HRP-conjugated secondary (2 ) antibodies bind the Fcregion of the 1 antibody. Through this binding, the HRP enzymeconjugated to the 2 antibody can catalyze a colorimetric reaction uponsubstrate addition. Due to the qualitative nature of the Cell-Based ELISA,multiple normalization methods are described: 1) a monoclonal antibodyspecific for human GAPDH is included to serve as an internal positivecontrol in normalizing the target absorbance values. 2) Following thecolorimetric measurement of HRP activity via substrate addition, the CrystalViolet whole-cell staining method is used to determine cell density. Afterstaining, the results can be analyzed by normalizing the absorbance valuesto cell amounts, by which the plating difference can be adjusted. 3) If aphosphorylated target is being detected, an antibody against the nonphosphorylated counterpart will be provided for normalization purposes.The absorbance values obtained for the non-phosphorylated target can beused to normalize the absorbance values for the phosphorylated target.INOS Colorimetric Cell-Based ELISAThe INOS Cell-Based ELISA Kit is a convenient, lysate-free, highthroughput and sensitive assay kit that can monitor INOS proteinexpression profile in cells. The kit can be used for measuring the relativeamounts of INOS in cultured cells as well as screening for the effects thatvarious treatments, inhibitors (ie. siRNA or chemicals), or activators haveon INOS.LSBio INOS 3

ASSAY FORMATLSBio INOS 4

ASSAY RESTRICTIONS This ELISA kit is intended for research purposes only, NOTdiagnostic or clinical procedures of any kind. Materials included in this kit should NOT be used past the expirationdate on the kit label. Reagents or substrates included in this kit should NOT be mixed orsubstituted with reagents or substrates from any other kits. Variations in pipetting technique, washing technique, operatorlaboratory technique, kit age, incubation time or temperature maycause differences in binding affinity of the materials provided. The assay is designed to eliminate interference and background byother cellular macromolecules or factors present within any biologicalsamples. However, the possibility of background noise cannot be fullyexcluded until all factors have been tested using the assay kit.LSBio INOS 5

ANTIBODY SPECIFICITYAnti-INOS AntibodyThe Anti-INOS Antibody is a rabbit polyclonal antibody. It was tested onWestern Blots for specificity. The data in Figure 2 shows that a singleprotein band was detected. This protein band can be blocked by thesynthesized immunogen peptide.Figure 2. Western blot analysis of extracts from NIH-3T3 cells, usingiNOS (Ab-151) Antibody.LSBio INOS 6

Anti-GAPDH AntibodyThe Anti-GAPDH Antibody is a mouse monoclonal antibody. It was testedon Western Blots with the tissue lysates from human, mouse, and rat forspecificity. The data in Figure 3 shows that a single protein band wasdetected from all three lysates.Figure 3. Western blot analysis of tissue lysates from human (1),mouse (2) and rat (3).LSBio INOS 7

MATERIALS INCLUDEDReagent96-Well Cell CultureClear-Bottom Microplate10x TBSQuenching BufferBlocking Buffer15x Wash Buffer100x Anti-INOS Antibody(Rabbit Polyclonal)100x Anti-GAPDH Antibody(Mouse Monoclonal)HRP-ConjugatedAnti-Rabbit IgG AntibodyHRP-ConjugatedAnti-Mouse IgG AntibodyPrimary Antibody DiluentReady-to-Use SubstrateStop SolutionCrystal Violet SolutionSDS SolutionAdhesive Plate SealsQuantityContainer1 Plate-24 ml (10x)24 ml (1x)50 ml (1x)50 ml (15x)ClearClearClearClear60 µl (100x)Purple60 µl (100x)Green6 ml (1x)Glass6 ml (1x)Glass12 ml (1x)12 ml (1x)12 ml (1x)6 ml (1x)24 ml (1x)4 SealsClearBrownClearGlassClear-STORAGE AND STABILITYUpon receipt, the kit should be stored at 4 C. The un-opened kit will bestable for up to 6 months from the date of shipment if stored at 4 C. DilutedAnti-INOS Antibody and diluted Anti-GAPDH Antibody can each be storedat 4 C for up to two weeks. HRP-Conjugated Anti-Rabbit IgG Antibody andHRP-Conjugated Anti-Mouse IgG Antibody will be stable at 4 C for up tosix months. The SDS Solution should be stored at room temperature orwarmed up to room temperature if stored at 4 C.LSBio INOS 8

BUFFER PREPARATION AND RECOMMENDATIONSNote: Please remember to allow all solutions to warm up to roomtemperature prior to use.1x TBS – 1x TBS is used to wash cells seeded on the plate. 1x TBS can beprepared by adding 1 volume of 10x TBS provided in the kit to 9 volumes ofddH2O.Fixing Solution – This solution is NOT provided. Fixing Solution is used tofix cells after cell culture. It is prepared by adding formaldehyde to 1x TBSwith light mixing. The 4% formaldehyde is used for adherent cells and 8%formaldehyde is used for suspension cells and loosely attached cells. 37%formaldehyde can be purchased from Sigma Cat# F-8775.Quenching Buffer – This solution is provided as ready-to-use. QuenchingBuffer is used to inactivate the endogenous peroxidase activity of theseeded cells.Blocking Buffer – This solution is provided as ready-to-use. BlockingBuffer is used to block additional binding sites in each well.Wash Buffer – This buffer is provided as a 15x solution. 1x Wash Buffercan be prepared by adding 1 volume of 15x Wash Buffer provided in the kitto 14 volumes of ddH2O.100x Anti-INOS Antibody – This antibody is a rabbit polyclonal antibody.This antibody was tested to be specific for the INOS protein. The suppliedantibody is a 100x solution. Make 1:100 dilutions in Primary AntibodyDiluent prior to use. The diluted primary antibody can be stored at 4 C forup to two weeks.100x Anti-GAPDH Antibody – This antibody is a mouse monoclonalantibody. This antibody was tested to be specific for GAPDH. The suppliedantibody is a 100x solution. Make 1:100 dilutions in Primary AntibodyDiluent prior to use. The diluted primary antibody can be stored at 4 C forup to two weeks.LSBio INOS 9

HRP-Conjugated Anti-Rabbit IgG Antibody – This solution is provided asready-to-use. HRP-Conjugated Anti-Rabbit IgG Antibody is used as thesecondary antibody to detect the target-bound, primary rabbit antibodies.HRP-Conjugated Anti-Mouse IgG Antibody – This solution is provided asready-to-use. HRP-Conjugated Anti-Mouse IgG Antibody is used as thesecondary antibody to detect the target-bound, primary mouse antibodies.Primary Antibody Diluent – This solution is provided as ready-to-use. Usethis solution to dilute the provided antibodies.Ready-to-Use Substrate – This solution is provided as ready-to-use.Ready-to-Use Substrate must be warmed to room temperature before use.Keep away from light as this solution is light-sensitive.Stop Solution – This solution is provided as ready-to-use. Stop Solutionmust be handled with caution as it contains 2 N Sulfuric Acid (H2SO4) andis corrosive. Wear eye protection and gloves when handling.Crystal Violet Solution – This solution is provided as ready-to-use. CrystalViolet is an intense stain used to stain cell nuclei. Avoid contact with skinand clothing.SDS Solution – This solution is provided as ready-to-use. SDS is used tosolubilize the Crystal Violet in preparation for cell staining. Store thissolution at room temperature or warm up to room temperature if stored at4 C.ADDITIONAL MATERIALS REQUIREDLSBio INOS 10

The following materials and equipment are NOT provided in this kit but arenecessary to successfully conduct the experiment: Microplate reader able to measure absorbance at 450 nm and/or 595nm for Crystal Violet Cell Staining (Optional) Micropipettes with capability of measuring volumes ranging from 1 μlto 1 ml 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from othersources Deionized or sterile water Squirt bottle, manifold dispenser, multichannel pipette reservoir orautomated microplate washer Graph paper or computer software capable of generating ordisplaying logarithmic functions Absorbent papers or vacuum aspirator Test tubes or microfuge tubes capable of storing 1 ml Orbital shaker Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)HEALTH AND SAFETY PRECAUTIONS Reagents provided in this kit may be harmful if ingested, inhaled orabsorbed through the skin. Fixing Solution contains formaldehyde. Formaldehyde is known to bea highly toxic reagent. Personal protection is strongly recommendedwhile working with this chemical. Stop Solution contains 2 N Sulfuric Acid (H2SO4) and is an extremelycorrosive agent. Please wear proper eye, hand and face protectionwhen handling this material. When the experiment is finished, be sureto rinse the plate with copious amounts of running water to dilute theStop Solution prior to disposing the plate or strips. Crystal Violet is an intense stain reagent. Avoid contact stain andclothing.LSBio INOS 11

EXPERIMENT DESIGN1) Cell Line: The cell line must express the target protein. This protocolcan be used directly for adherent cells. For suspension cells andloosely attached cells, two steps are required: 1) Coat the plates with100 µl of 10 µg/ml Poly-L-Lysine (Sigma Cat# P4832, not included) toeach well of the 96-well plate for 30 minutes at 37 C beforeproceeding to Step 1 of Assay Protocol (on page 13). Use 8%formaldehyde to fix the cells on Step 5 of Assay Protocol.2) Cell Number and Sensitivity: The number of cells plated onto the96-well plates depends on the expression level of INOS protein in thecells, cell size, treatment conditions and incubation time. The cellsused for testing should be around 75-90% confluent. Typically forHeLa cells, seed 30,000 cells per well overnight for treatment thefollowing day. The INOS Colorimetric Cell-Based ELISA Kit candetect INOS expression in as little as 5,000 HeLa cells.3) Cell Treatment: The cells can be treated with inhibitors, activators,stimulators (ie. chemicals, proteins/peptides) or a combination of thesubstances listed above. The cells can be treated with UV and serumstarvation to meet the needs of the end-user.4) Positive and Negative Controls: Mouse Anti-GAPDH Antibody(included) should be used to detect the internal positive controls fornormalization of OD values of the target protein. The negativecontrols are HRP-Conjugated Anti-Rabbit IgG Antibody and HRPConjugated Anti-Mouse IgG Antibody alone in different wells (withoutthe primary antibodies). Both positive and negative controls should beperformed in the same plate with the INOS target experiments.5) Accuracy and Precision: Each condition should be performed induplicate or in triplicate.LSBio INOS 12

ASSAY PROTOCOLNote: Please read the whole manual before performing the experiment.1)Seed 200 µl of 20,000 adherent cells in culture medium in eachwell of a 96-well plate. The plates included in the kit are sterile andtreated for cell culture. For suspension cells and loosely attachedcells, coat the plates with 100 µl of 10 µg/ml Poly-L-Lysine (notincluded) to each well of a 96-well plate for 30 minutes at 37 Cprior to adding cells.2)Incubate the cells for overnight at 37 C, 5% CO2.3)Treat the cells as desired.4)Remove the cell culture medium and rinse with 200 µl of 1x TBS,twice.5)Fix the cells by incubating with 100 µl of Fixing Solution for 20minutes at room temperature. The 4% formaldehyde is used foradherent cells and 8% formaldehyde is used for suspension cellsand loosely attached cells. During the incubation, the platesshould be sealed with Parafilm. Note: Fixing Solution is volatile.Wear appropriate personal protection equipment (mask, glovesand glasses) when using this chemical.6)Remove the Fixing Solution and wash the plate 3 times with 200 µl1x Wash Buffer for five minutes each time with gentle shaking onthe orbital shaker. The plate can be stored at 4 C for a week.Note: For all wash steps, tap the plate gently on absorbentpapers to remove the solution completely.7)Add 100 µl Quenching Buffer and incubate for 20 minutes at roomtemperature.8)Wash the plate 3 times with 1x Wash Buffer for 5 minutes at atime, with gentle shaking on the shaker.9)Add 200 µl of Blocking Buffer and incubate for 1 hour at roomtemperature.LSBio INOS 13

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