Sandwich ELISA protocol Use this workflow when a target is captured by one antibody and detected by a second antibody that recognizes a different epitope. Use this format when: Quantifying soluble proteins, cytokines, hormones, histones, modified proteins, or other targets where a matched antibody pair is available.Optimize first: Capture antibody concentration, detection antibody concentration, sample dilution, blocking buffer, wash stringency, and standard curve range. 1 Prepare plate map and reagentsPlan standards, blanks, negative controls, positive controls, and samples before starting. Bring coated plate, standards, samples, wash buffer, blocking buffer, detection antibody, enzyme conjugate, substrate, and stop solution to room temperature unless the kit manual says otherwise. For manual coating, use a high-binding 96-well ELISA plate. 2 Coat capture antibodyDilute capture antibody in carbonate-bicarbonate coating buffer, pH 9.6, or the buffer specified for the antibody pair. Add 100 uL per well. Cover the plate and incubate overnight at 4 degrees C, or 2 hours at room temperature as a faster starting condition. Aspirate the coating solution after incubation. 3 Wash and blockWash 3 times with 250-300 uL per well 1x PBST or TBST. Add 200 uL per well blocking buffer, such as 1-5 percent BSA, casein, or nonfat dry milk in PBS/TBS. Incubate 1 hour at room temperature. Wash 3 times. 4 Add standards and samplesPrepare standards in duplicate or triplicate using the recommended diluent. Dilute samples so readings fall within the linear range. Add 100 uL per well and incubate 1-2 hours at room temperature, or overnight at 4 degrees C for low-abundance targets. Wash 4-5 times. 5 Add detection antibodyDilute detection antibody in assay diluent. If this product is used as the detection antibody, add Histone H2AX Monoclonal Antibody at the datasheet-recommended dilution or start at 0.25-2 ug/mL for optimization. Add 100 uL per well and incubate 1 hour at room temperature. Wash 4-5 times. 6 Add enzyme conjugate and substrateAdd 100 uL per well HRP-conjugated secondary antibody or streptavidin-HRP as required by the detection antibody system. Incubate 30-60 minutes at room temperature protected from light when needed. Wash 5 times, then add 100 uL TMB substrate per well. 7 Stop and readDevelop color for 5-20 minutes until the standard curve is visible but not saturated. Add 50-100 uL stop solution per well and read absorbance at 450 nm, with 570 or 620 nm correction if available. Analyze standards with a 4-parameter logistic curve when appropriate. ControlsInclude blank wells, zero standard, standard curve, matrix control, no-sample control, and positive sample when available.Wash consistencyMost high background in sandwich ELISA comes from insufficient washing, over-concentrated detection antibody, or incompatible blocking buffer.
Indirect ELISA protocol Use this workflow to detect antibody binding to an immobilized antigen or target molecule using an unconjugated primary antibody and labeled secondary antibody. Use this format when: Testing antigen recognition, screening antibody reactivity, comparing sample antibody levels, or using an unconjugated primary antibody.Optimize first: Coating concentration, sample or antibody dilution, blocking buffer, secondary antibody dilution, and wash stringency. 1 Coat antigen or targetDilute purified antigen, peptide, protein, histone, modified target, cell lysate, or other coating material in carbonate-bicarbonate buffer or PBS. Add 100 uL per well. A common starting range is 0.5-5 ug/mL for purified protein or peptide. Incubate overnight at 4 degrees C or 2 hours at room temperature. 2 Wash and blockAspirate coating solution and wash 3 times with 250-300 uL per well PBST or TBST. Add 200 uL per well blocking buffer. Incubate 1 hour at room temperature, then wash 3 times. 3 Add primary antibodyDilute Histone H2AX Monoclonal Antibody in assay diluent. Start with the datasheet-recommended dilution when available; otherwise test a dilution series such as 1:250, 1:500, 1:1000, and 1:2000. Add 100 uL per well and incubate 1 hour at room temperature or overnight at 4 degrees C for weak targets. 4 Wash after primary antibodyWash 4-5 times with 250-300 uL per well wash buffer. Tap the inverted plate on clean absorbent material after the final wash to remove residual liquid without drying the wells. 5 Add enzyme-conjugated secondary antibodyDilute HRP- or AP-conjugated secondary antibody in assay diluent, commonly 1:2000-1:10000 depending on supplier and signal strength. Add 100 uL per well and incubate 30-60 minutes at room temperature. 6 Develop signalWash 5 times. Add 100 uL substrate per well. For HRP/TMB, develop 5-20 minutes protected from strong light, then stop with 50-100 uL stop solution and read at 450 nm. 7 Interpret resultsSubtract blank background and compare signal across antigen-coated, uncoated, no-primary, and no-secondary controls. High signal in no-primary wells suggests secondary antibody or blocking-related background. Antigen coatingToo much coated antigen can increase background. If the signal is high but nonspecific, reduce coating concentration before changing everything else.Primary antibody variableThe product page can replace the primary antibody variable in this protocol with the selected antibody name.
Direct ELISA protocol Use this workflow when the target is immobilized or captured and detected directly by a labeled antibody, direct detection reagent, or kit-specific detection system. Use this format when: A direct detection kit, directly labeled antibody, or target-specific direct detection reagent is available.Optimize first: Coating or sample input, detection reagent amount, incubation time, wash stringency, and substrate development time. 1 Prepare plate and samplesPlan standards, blanks, negative controls, positive controls, and sample dilutions. Add 100 uL per well of standards or samples to the assay plate according to the kit format or coating requirement. 2 Immobilize target if requiredFor coated-plate direct ELISA, incubate antigen or sample in the well overnight at 4 degrees C or 2 hours at room temperature. For direct target-detection kits, follow the kit manual for binding, capture, or sample incubation conditions. 3 Wash and blockWash 3 times with 250-300 uL per well wash buffer. Add 200 uL per well blocking buffer for 30-60 minutes if the assay format requires blocking. Some direct detection kits include a proprietary blocking or assay buffer that should be used instead. 4 Add direct detection reagentAdd 100 uL per well of the directly labeled antibody, direct detection reagent, or enzyme-conjugated target-specific reagent. If using a directly conjugated antibody, start with the datasheet-recommended dilution or test 0.1-1 ug/mL. Incubate 30-90 minutes at room temperature protected from light when appropriate. 5 Wash and developWash 4-5 times to reduce background. Add 100 uL per well substrate. Develop until standards or positive controls show clear separation without saturation. 6 Stop and readAdd 50-100 uL stop solution when using TMB and read at 450 nm. Use the correction wavelength recommended for the plate reader if available. Compare results to the standard curve or kit-specific calculation method. Direct detection kitsSome EpigenTek assay kits use direct target-detection formats for small molecules, DNA/RNA damage markers, or epigenetic modifications. In those cases, the kit manual overrides generic antibody-only workflow assumptions.Background controlInclude blank, no-target, no-detection-reagent, and positive control wells when compatible with the kit or assay design.
Competitive ELISA protocol Use this workflow when sample target competes with immobilized or labeled target for antibody or binding reagent. Use this format when: The analyte is small, has one dominant epitope, or is measured by competition rather than two-antibody sandwich capture.Optimize first: Sample dilution, competitor concentration, antibody concentration, incubation time, and standard curve range. 1 Prepare standards and samplesPrepare a full standard curve using the assay diluent recommended for the target. Dilute samples into the same matrix when possible. Competitive assays often need careful sample dilution because high target concentration produces lower signal. 2 Add standards, samples, and competitorAdd 50-100 uL per well of standards or samples according to the assay design. Add competitor, tracer, or labeled target reagent if the protocol requires a pre-mix. Mix gently without splashing. 3 Add antibody or binding reagentAdd the antibody or target-specific binding reagent at the optimized concentration. When using an antibody-based format, add Histone H2AX Monoclonal Antibody only if the product is the intended competitive binding antibody and follow the datasheet dilution first. 4 Incubate competition reactionIncubate 1-2 hours at room temperature, or as specified by the kit manual. Keep incubation times consistent across the plate because competitive assays are especially sensitive to timing differences. 5 Wash thoroughlyWash 4-5 times with 250-300 uL per well wash buffer. Avoid drying the wells. Inconsistent washing can distort the inverse standard curve. 6 Develop and readAdd 100 uL substrate per well and develop until the low-analyte standards produce strong but unsaturated signal. Stop and read at the appropriate wavelength. Fit the data using the curve model recommended by the kit or assay format. Signal directionIn competitive ELISA, stronger sample target usually means lower signal. Label graphs and reports clearly to avoid reversed interpretation.TimingUse a multichannel pipette and consistent timing between rows when possible.
Cross-linking ChIP protocol Use cross-linking ChIP for transcription factors, cofactors, chromatin regulators, and many DNA-associated proteins that may not remain bound during native extraction. Use this format when: The target is a non-histone protein, a weak or transient DNA-binding factor, or a chromatin complex that benefits from formaldehyde stabilization. Relevant ChIP kits: For standard mammalian cell ChIP, see the EpiQuik Chromatin Immunoprecipitation (ChIP) Kit. For low-input or high-sensitivity ChIP from cells or tissues, see the ChromaFlash High-Sensitivity ChIP Kit. Optimize first: Crosslinking time, sonication conditions, antibody amount, bead type, wash stringency, and qPCR primer performance. 1Prepare cells and fresh buffersStart with 1 x 106 to 1 x 107 cells per IP. Chill PBS, wash buffers, and collection tubes. Add fresh protease inhibitors to all lysis and wash buffers immediately before use. 2Crosslink protein-DNA complexesAdd formaldehyde directly to culture medium to 1 percent final concentration. Incubate 10 minutes at room temperature with gentle rocking. For sensitive epitopes, test 5, 8, and 10 minutes in a pilot experiment. 3Quench crosslinkingAdd glycine to 125 mM final concentration and incubate 5 minutes at room temperature. Aspirate medium and wash cells 2-3 times with cold PBS containing protease inhibitors. 4Collect and lyse cellsScrape adherent cells in 1 mL cold PBS per 10 cm dish or pellet suspension cells at 500 x g for 5 minutes at 4°C. Resuspend the pellet in ChIP lysis buffer at about 1 mL per 1 x 107 cells. Incubate 10-15 minutes on ice. 5Fragment chromatinSonicate on ice or in a cooled sonication system to a starting DNA size of 200-800 bp for ChIP-qPCR or 150-500 bp for many ChIP-seq workflows. Keep samples cold and avoid foaming. Clarify at 12,000 x g for 10 minutes at 4°C. 6Check fragmentation and save inputRemove 20-50 uL sheared chromatin for reversal and gel or Bioanalyzer check. Save 1-10 percent input chromatin before antibody addition. Store input at 4°C overnight or at -20°C if processing later. 7Pre-clear chromatinPre-clear chromatin with 20-40 uL blocked protein A/G magnetic beads for 30-60 minutes at 4°C with rotation. Transfer the supernatant to a new tube and avoid carrying over beads. 8Add ChIP antibodyAdd 2-5 ug Histone H2AX Monoclonal Antibody per IP as a starting range. For low-abundance targets, test 5-10 ug or more chromatin. Include IgG control and a known positive ChIP antibody when available. Rotate overnight at 4°C. 9Capture immune complexesAdd 30-50 uL washed, blocked protein A/G magnetic beads. Rotate 2-4 hours at 4°C. Place tubes on a magnetic stand and remove unbound supernatant. 10Wash beads sequentiallyWash beads with 1 mL cold buffer per wash. Use 2 washes low-salt buffer, 2 washes high-salt buffer, 1 wash LiCl buffer, and 2 washes TE buffer as a stringent starting sequence. Rotate 3-5 minutes per wash unless the target is fragile. 11Elute and reverse crosslinksElute twice with 100-120 uL elution buffer per elution at room temperature for 10-15 minutes. Combine eluates. Add NaCl to 200 mM final concentration and reverse crosslinks at 65°C for 4 hours to overnight. Treat with RNase A and proteinase K before DNA purification. 12Purify and analyze DNAPurify DNA using a spin-column, magnetic bead, or phenol/chloroform cleanup method compatible with low DNA input. Elute in 30-50 uL low-EDTA TE or nuclease-free water. Analyze enrichment by qPCR or proceed to sequencing library preparation. Fragment sizeLarge fragments can reduce resolution. Over-shearing can reduce recovery or damage epitopes. Always verify shearing before interpreting poor enrichment.CrosslinkingOver-crosslinking can mask epitopes and lower antibody capture. Under-crosslinking can lose weakly bound targets.
Native ChIP protocol Use native ChIP for histones and many histone modifications when preserving native nucleosome structure is preferred and formaldehyde crosslinking is not required. Use this format when: The target is a histone protein or histone modification with strong chromatin association. Optimize first: MNase digestion, nucleosome fragment range, antibody amount, and salt conditions during immunoprecipitation. 1Collect cells without crosslinkingStart with 1 x 106 to 5 x 106 cells per IP for histone targets as a practical starting range. Wash cells twice with cold PBS and keep all steps cold unless digestion conditions require warmth. 2Prepare nucleiResuspend the cell pellet in hypotonic or nuclear isolation buffer containing protease inhibitors. Incubate on ice 10 minutes. Dounce gently if needed. Pellet nuclei at 800-1,000 x g for 5 minutes at 4°C. 3Digest chromatin with MNaseResuspend nuclei in MNase digestion buffer containing calcium. Add a titrated amount of micrococcal nuclease and incubate at 37°C for 5-20 minutes. Start with a small pilot digestion to identify conditions that produce mostly mono- and oligonucleosomes. 4Stop digestionAdd EDTA to 5-10 mM final concentration to stop MNase activity. Place samples on ice immediately. Avoid prolonged digestion after the stop step. 5Release soluble chromatinLyse nuclei gently with native ChIP lysis or IP buffer containing 0.1-0.5 percent NP-40 or Triton X-100 and physiological salt. Incubate on ice 10 minutes, then clarify at 12,000 x g for 10 minutes at 4°C. 6Check nucleosome size and save inputRemove 20-50 uL chromatin for DNA purification and size check. A useful native ChIP digestion often includes mono-, di-, and tri-nucleosome DNA with a major range around 150-600 bp. Save 1-10 percent input. 7Pre-clear chromatinIncubate chromatin with 20-40 uL blocked protein A/G beads for 30-60 minutes at 4°C with rotation. Transfer the pre-cleared supernatant to a new tube. 8Add histone or PTM antibodyAdd 1-5 ug Histone H2AX Monoclonal Antibody per IP as a starting range. For abundant histone marks, lower antibody and lower chromatin input may be sufficient. Rotate 2-4 hours or overnight at 4°C. 9Capture with beadsAdd 30-50 uL washed, blocked protein A/G beads and rotate 1-2 hours at 4°C. Use magnetic separation or low-speed spin collection depending on bead type. 10Wash under native conditionsWash 4-6 times with cold native ChIP wash buffer. Start with 1 mL per wash and 3-5 minutes rotation. Increase salt or detergent only if background is high and recovery remains acceptable. 11Elute DNA-protein complexesElute with 100-200 uL elution buffer compatible with DNA recovery, commonly SDS-containing elution buffer or proteinase K digestion buffer. Treat with proteinase K at 55-65°C for 1-2 hours. 12Purify DNA and quantify enrichmentPurify DNA and elute in 30-50 uL. Analyze by qPCR using input-normalized percent input or fold enrichment over IgG. For sequencing, confirm DNA yield and library compatibility before pooling samples. Best fitNative ChIP is usually strongest for histone marks and may not preserve weak or transient transcription factor binding.MNase titrationMNase digestion is sample dependent. Always optimize enzyme amount and digestion time before large experiments.
ChIP-qPCR analysis protocol Use ChIP-qPCR to validate enrichment at defined genomic loci after native or cross-linking ChIP. Use this format when: Testing a known locus, validating ChIP enrichment, optimizing a new ChIP antibody, or confirming ChIP-seq findings. Relevant ChIP-qPCR kits: Pair ChIP-enriched DNA with the EpiQuik Quantitative PCR Fast Kit for fast quantitative real-time PCR analysis of ChIP DNA. Use a ChIP enrichment kit such as EpiQuik ChIP Kit or ChromaFlash High-Sensitivity ChIP Kit upstream. Optimize first: Primer specificity, amplicon size, qPCR efficiency, positive and negative loci, input dilution, and replicate consistency. 1Design ChIP-qPCR primersDesign primers for 70-150 bp amplicons centered on the expected binding or modification region. Include at least one positive locus and one negative locus when possible. 2Validate primer efficiencyTest primers on input DNA dilution series. Aim for a single melt peak and similar amplification efficiency across primer sets. Redesign primers if primer dimers or non-specific products appear. 3Dilute ChIP and input DNAUse 1-2 uL purified ChIP DNA per 10-20 uL qPCR reaction as a starting point. Dilute input DNA so Ct values fall in the linear range and are not several cycles earlier than the ChIP samples. 4Set up qPCR reactionsFor each 20 uL reaction, combine 10 uL 2x qPCR master mix, primer mix at validated concentration, 1-2 uL DNA, and nuclease-free water. The EpiQuik Quantitative PCR Fast Kit can be used for ChIP DNA qPCR analysis. Run technical duplicates or triplicates for input, target IP, IgG IP, and controls. 5Use consistent controlsInclude no-template controls, input DNA, IgG IP DNA, positive control IP DNA when available, and negative genomic loci. If using Histone H2AX Monoclonal Antibody for a new target, include a known responsive biological condition when possible. 6Calculate percent inputCorrect input Ct for dilution, then calculate percent input for each IP. A common formula is 100 x 2^(adjusted input Ct - IP Ct). Apply the same formula to target IP and IgG IP. 7Calculate fold enrichmentCalculate enrichment over IgG or over a negative locus when appropriate. Report the normalization method clearly because percent input and fold enrichment answer different questions. 8Interpret enrichment with controlsA useful ChIP-qPCR result should show enrichment at expected loci, low IgG background, acceptable input amplification, and reproducibility across biological replicates. Weak signal at all loci usually points to chromatin, antibody, or wash stringency issues. Amplicon sizeShort amplicons perform better with fragmented ChIP DNA. Long amplicons can underperform after heavy shearing.Negative lociNegative loci help distinguish real locus-specific enrichment from antibody or bead background.
ChIP-seq preparation and QC protocol Use this workflow after native or cross-linking ChIP when the enriched DNA will be used for sequencing library preparation. Use this format when: ChIP DNA will be converted into sequencing libraries for genome-wide mapping. Relevant ChIP-seq kit: For an all-in-one ChIP and ChIP-seq library preparation workflow, see the EpiNext ChIP-Seq High-Sensitivity Kit (Illumina). Optimize first: Fragment size, DNA recovery, adapter dilution, PCR cycle number, duplicate rate, and positive or negative qPCR QC before sequencing. 1Confirm ChIP enrichment before library prepRun ChIP-qPCR on at least one positive and one negative locus before committing samples to sequencing. Proceed when target IP shows clear enrichment over IgG and negative loci. 2Quantify ChIP DNA carefullyUse a sensitive fluorometric method or library prep kit guidance for low-input DNA. ChIP DNA may be below the accurate range of standard absorbance methods. 3Assess fragment sizeCheck purified ChIP DNA or library intermediates by Bioanalyzer, TapeStation, Fragment Analyzer, or gel-based method. Many ChIP-seq workflows use enriched DNA around 150-500 bp before library amplification. 4Prepare library with low-input conditionsUse a ChIP-seq compatible low-input DNA library kit such as the EpiNext ChIP-Seq High-Sensitivity Kit (Illumina). Follow kit-specific end repair, A-tailing, adapter ligation, cleanup, and PCR conditions. Dilute adapters when DNA input is low to reduce adapter dimers. 5Optimize PCR cycle numberUse the fewest PCR cycles that produce enough library for sequencing. Excess PCR can increase duplicate reads, skew enrichment, and create library artifacts. 6Clean up and size select libraryUse magnetic bead cleanup or kit-recommended size selection to remove primers, enzymes, salts, and adapter dimers. Verify final library size distribution before pooling. 7Quantify final libraryQuantify libraries using qPCR-based library quantification or another sequencing-compatible method. Normalize samples before pooling. 8Review sequencing designInclude biological replicates, matching input controls, and IgG controls when informative. For broad histone marks, plan sufficient depth and appropriate peak-calling settings. For narrow transcription factor peaks, prioritize enrichment and replicate consistency. Library artifactsAdapter dimer and high PCR duplication often indicate too little input DNA, excess adapter, or too many PCR cycles.ReplicatesBiological replicate consistency is more useful than a single high-depth library with poor ChIP enrichment.
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