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Base Cat #A74565

NDUFS4 Recombinant Monoclonal Antibody [9H1]Recombinant

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Profiles NDUFS4 within target biology and pathway-focused research.

Application: ELISAFCIHC
Clonality: Monoclonal
Conjugate: Unconjugated
Host: HEK293F Cell
Isotype: Rabbit IgG
Purification: Affinity Chromatography
Reactivity: Human
Recombinant: Yes
100% Guarantee: 6 months
Application Guarantee for AntibodiesRecombinant Rabbit Monoclonal Abs
Catalog No.SizePriceQty
A74565-100100µl $299.00 
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Product Overview

Background
Accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I), that is believed not to be involved in catalysis. Complex I functions in the transfer of electrons from NADH to the respiratory chain. The immediate electron acceptor for the enzyme is believed to be ubiquinone.

Description
NDUFS4 Recombinant Monoclonal Antibody [9H1]. Unconjugated. Raised in: HEK293F Cell.

Formulation
Buffer: Rabbit IgG in phosphate buffered saline, pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol.

Specificity
Human

Isotype
Rabbit IgG

Uniprot ID
O43181

Purification
Affinity-chromatography

Immunogen
A synthesized peptide derived from human NDUFS4

Storage
Upon receipt, store at -20°C or -80°C. Avoid repeated freeze.

Alternative Names
NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mitochondrial (Complex I-18 kDa) (CI-18 kDa) (Complex I-AQDQ) (CI-AQDQ) (NADH-ubiquinone oxidoreductase 18 kDa subunit), NDUFS4

Application
ELISA, IHC, FC; Recommended dilution: IHC:1:50-1:200, FC:1:50-1:200

Datasheet & SDS

PDF
Datasheet/User Guide: A74565 If the above file is not available online, you can request a copy by contacting us.

Applications

Suggested protocols for NDUFS4 Recombinant Monoclonal Antibody [9H1] should be self-optimized according to the user's experiment.

Choose Your IHC Workflow

Use this immunohistochemistry protocol guide to run antibody staining in FFPE tissue, frozen tissue, fluorescent tissue staining, or optimization workflows.

FFPE chromogenic IHC protocol

Use this workflow for formalin-fixed paraffin-embedded tissue stained with HRP/DAB or another chromogenic detection system.

  • Use this format when: Evaluating protein expression or localization in FFPE tissue sections by brightfield microscopy.
  • Optimize first: Antigen retrieval buffer, retrieval time, primary antibody dilution, blocking, detection chemistry, and hematoxylin counterstain.
1
Bake and deparaffinize slides
Bake 4-5 um FFPE sections at 60 degrees C for 30-60 minutes. Deparaffinize in xylene, 2 changes for 5 minutes each. Rehydrate through 100 percent, 95 percent, and 70 percent ethanol, 2-3 minutes each, then rinse in water.
2
Perform antigen retrieval
Place slides in citrate buffer pH 6.0 or Tris-EDTA buffer pH 9.0. Heat near boiling for 10-20 minutes using a pressure cooker, microwave, steamer, or water bath. Cool slides in buffer for 20 minutes, then rinse in TBS or PBS.
3
Block endogenous activity
For HRP detection, incubate slides in 3 percent hydrogen peroxide for 10 minutes to quench endogenous peroxidase. Rinse 3 times in buffer.
4
Block nonspecific binding
Apply protein block, 5 percent normal serum, or 1-5 percent BSA for 20-60 minutes at room temperature. Drain but do not rinse if the detection system recommends leaving blocking protein on the section.
5
Add primary antibody
Dilute NDUFS4 Recombinant Monoclonal Antibody [9H1] in antibody diluent. Start with the datasheet-recommended IHC dilution or test 1:50, 1:100, 1:250, and 1:500. Apply 100-200 uL per section and incubate 1 hour at room temperature or overnight at 4 degrees C in a humidified chamber.
6
Wash after primary antibody
Wash 3 times for 5 minutes each in TBS-T or PBS-T. Keep slides covered with buffer and do not let tissue dry.
7
Apply secondary or polymer detection
Apply HRP polymer, biotinylated secondary plus streptavidin-HRP, or another detection reagent according to the system instructions. Typical incubation is 20-30 minutes at room temperature. Wash 3 times.
8
Develop chromogen
Apply DAB or other chromogen for 1-10 minutes while monitoring signal under a microscope. Stop development by rinsing in water.
9
Counterstain and mount
Counterstain with hematoxylin for 15 seconds to 2 minutes, blue in running tap water or bluing reagent, dehydrate through ethanol, clear in xylene, and mount with permanent mounting medium.
Retrieval optimizationIf signal is weak, compare pH 6 and pH 9 retrieval before dramatically increasing antibody concentration.
Negative controlUse no-primary, isotype control when appropriate, and known negative tissue to evaluate nonspecific staining.

Frozen tissue IHC protocol

Use this workflow for frozen sections when antigen preservation is more important than FFPE morphology.

  • Use this format when: The epitope is fixation-sensitive, lipid-rich tissue is used, or frozen-section staining is preferred.
  • Optimize first: Fixation method, section thickness, drying time, blocking, primary antibody dilution, and tissue autofluorescence or endogenous enzyme activity.
1
Prepare frozen sections
Cut 5-10 um cryosections and mount onto charged slides. Air dry 10-30 minutes at room temperature. Store slides cold if staining later.
2
Fix sections
Fix with cold acetone for 10 minutes at -20 degrees C, cold methanol for 5-10 minutes, or 4 percent paraformaldehyde for 10-15 minutes depending on the target. Rinse gently in PBS or TBS.
3
Block endogenous activity if needed
For HRP-based detection, quench endogenous peroxidase with 0.3-3 percent hydrogen peroxide for 10 minutes. For biotin-based detection, consider avidin/biotin blocking when tissue has high endogenous biotin.
4
Block nonspecific binding
Block 20-60 minutes with 5 percent normal serum or 1-5 percent BSA in PBS/TBS. Use serum from the secondary antibody host species when practical.
5
Add primary antibody
Dilute NDUFS4 Recombinant Monoclonal Antibody [9H1] in antibody diluent. Start with the datasheet-recommended frozen IHC dilution or test 1:50 to 1:500. Apply 100-200 uL per section and incubate 1 hour at room temperature or overnight at 4 degrees C.
6
Wash and detect
Wash 3 times for 5 minutes. Apply secondary antibody, polymer detection, or fluorescent secondary antibody according to the detection system. Incubate 30-60 minutes.
7
Develop or mount
For chromogenic detection, apply substrate until signal develops, rinse, counterstain, and mount. For fluorescent detection, protect from light, counterstain with DAPI if needed, and mount with antifade medium.
MorphologyFrozen sections usually preserve antigenicity better but morphology can be less crisp than FFPE tissue.
Section adhesionUse charged slides and avoid harsh washing if sections lift from the slide.

Fluorescent IHC protocol

Use this workflow for fluorescent staining of tissue sections when colocalization or multiplex imaging is needed.

  • Use this format when: Tissue localization is needed with fluorescent readout, multiple markers, or confocal microscopy.
  • Optimize first: Autofluorescence control, antigen retrieval, fluorophore choice, antibody host species, and mounting medium.
1
Prepare and retrieve tissue
For FFPE tissue, deparaffinize, rehydrate, and perform antigen retrieval. For frozen tissue, fix according to target requirements. Rinse in PBS or TBS.
2
Reduce autofluorescence
Treat autofluorescent tissues with a compatible quenching reagent when needed. Avoid quenchers that interfere with the planned fluorophores.
3
Permeabilize and block
Use 0.1-0.3 percent Triton X-100 for intracellular access when compatible. Block with 5 percent normal serum or 1-5 percent BSA for 30-60 minutes.
4
Add primary antibody
Dilute NDUFS4 Recombinant Monoclonal Antibody [9H1] in blocking buffer. Start with the datasheet-recommended IHC/IF dilution or test 1:100, 1:250, and 1:500. Apply 100-200 uL per section and incubate 1-2 hours at room temperature or overnight at 4 degrees C.
5
Wash and add fluorescent secondary
Wash 3 times for 5 minutes. Add cross-adsorbed fluorescent secondary antibody at 1:500-1:1000 for 45-60 minutes protected from light.
6
Counterstain and mount
Wash 3 times. Add DAPI if desired. Mount with antifade medium and coverslip without bubbles.
7
Image and control
Acquire no-primary, single-color, and full-stain controls. Use identical image settings for samples that will be compared.
Multiplex tissue stainingFor multiple primary antibodies, confirm species compatibility or use directly conjugated primaries or sequential staining.
AutofluorescenceTissue autofluorescence can resemble true signal. Always compare to no-primary controls.

IHC optimization protocol

Use this workflow when an antibody or tissue type needs condition screening before final staining.

  • Use this format when: Signal is weak, background is high, staining is inconsistent, or the antibody is being validated in a new tissue.
  • Optimize first: Positive tissue, retrieval pH, antibody dilution, incubation time, detection strength, and blocking strategy.
1
Choose control tissue
Use a known positive tissue or cell pellet and a known negative tissue when available. Include the experimental tissue only after positive control staining is working.
2
Build a retrieval matrix
Test no retrieval, citrate pH 6.0, and Tris-EDTA pH 9.0 when tissue availability allows. Use the same section thickness and slide type for all conditions.
3
Test antibody dilution
Dilute NDUFS4 Recombinant Monoclonal Antibody [9H1] across a small matrix such as 1:50, 1:100, 1:250, and 1:500, or follow datasheet starting ranges. Keep retrieval and detection constant while testing dilution.
4
Compare incubation conditions
If signal is weak, compare 1 hour at room temperature with overnight at 4 degrees C. Longer incubation can improve weak signal but may increase background.
5
Adjust detection strength
If signal remains weak, increase polymer incubation within the detection system limits, use amplification, or extend chromogen development while monitoring background.
6
Reduce background
Increase wash time, reduce antibody concentration, change blocking buffer, shorten chromogen development, or add detergent to wash buffer if compatible with the tissue and detection system.
7
Lock the protocol
Once the best condition is chosen, stain all comparative samples together using the same retrieval, antibody dilution, incubation time, detection system, and development time.
One change at a timeChange one major variable at a time when troubleshooting so the cause of improvement is clear.
DocumentationRecord lot numbers, retrieval buffer, heating method, antibody dilution, incubation time, detection system, and imaging settings.
Open full IHC protocol

Choose Your Flow Cytometry Workflow

Use this flow cytometry protocol guide to run antibody staining for surface markers, intracellular targets, phospho-flow signaling, or multicolor panels.

Surface staining flow cytometry protocol

Use this workflow for extracellular antigens on live or fixed cells before acquisition by flow cytometry.

  • Use this format when: Detecting cell surface markers, receptors, lineage markers, or extracellular epitopes.
  • Optimize first: Cell viability, Fc blocking, antibody amount, staining volume, wash conditions, and compensation controls.
1
Prepare single-cell suspension
Collect cells and make a single-cell suspension. Filter clumps if needed. Count cells and transfer 0.5-1 x 10^6 cells per tube or well. Keep cells on ice or at 4 degrees C when staining sensitive surface markers.
2
Wash cells
Add 1-2 mL staining buffer per tube, or 150-200 uL per 96-well plate well. Staining buffer can be PBS or HBSS with 1-2 percent FBS or BSA and 1-2 mM EDTA. Centrifuge 300-500 x g for 5 minutes and discard supernatant.
3
Block Fc receptors
Resuspend cells in 50-100 uL staining buffer containing Fc block or 5-10 percent normal serum when working with Fc receptor-positive cells. Incubate 10-15 minutes at 4 degrees C.
4
Add antibody
Add NDUFS4 Recombinant Monoclonal Antibody [9H1] at the datasheet-recommended flow cytometry amount or start with 0.25-1 ug per 10^6 cells for unconjugated antibody. For conjugated antibodies, use the recommended test size or titrate 0.125x, 0.25x, 0.5x, 1x, and 2x. Final staining volume is commonly 50-100 uL.
5
Incubate
Incubate 20-30 minutes at 4 degrees C protected from light. Mix gently halfway through if cells settle.
6
Wash
Add staining buffer, centrifuge 300-500 x g for 5 minutes, and discard supernatant. Wash 2 times. Resuspend final pellet in 200-500 uL staining buffer for acquisition.
7
Add secondary antibody if needed
If using an unconjugated primary antibody, add fluorophore-conjugated secondary antibody after the first wash. Incubate 20-30 minutes at 4 degrees C protected from light, then wash 2 times.
8
Acquire data
Filter samples if clumps are present. Acquire enough events for the population of interest. Use viability dye, unstained cells, single-color controls, fluorescence minus one controls, and isotype controls when appropriate.
Antibody titrationFlow cytometry antibody concentration should be titrated. Too much antibody can increase background without improving separation.
TemperatureCold staining reduces internalization of some surface receptors.

Intracellular flow cytometry protocol

Use this workflow for intracellular proteins, transcription factors, cytokines, or nuclear targets after fixation and permeabilization.

  • Use this format when: The target is inside the cell or nucleus and requires membrane permeabilization.
  • Optimize first: Fixation buffer, permeabilization buffer, antibody dilution, cell loss during washes, and intracellular controls.
1
Prepare and optionally stain surface markers
Prepare 0.5-1 x 10^6 cells per tube or well. If surface markers are also needed, stain surface antigens first using the surface staining protocol before fixation unless the surface antibody is fixation-resistant.
2
Fix cells
Add fixation buffer according to the kit or reagent instructions. A common starting condition is 1-4 percent paraformaldehyde for 10-20 minutes at room temperature. Wash with staining buffer.
3
Permeabilize
Resuspend cells in permeabilization buffer, such as saponin-based buffer for many cytoplasmic targets or methanol/permeabilization kit buffer for nuclear and phospho targets. Incubate 10-30 minutes as recommended.
4
Block
Incubate cells in permeabilization buffer containing 1-5 percent serum or BSA for 10-20 minutes. Keep cells in permeabilization buffer during antibody incubation and washes when using saponin-based systems.
5
Add primary antibody
Dilute NDUFS4 Recombinant Monoclonal Antibody [9H1] in permeabilization buffer. Start with the datasheet-recommended flow dilution or titrate 0.25-2 ug per 10^6 cells. Incubate 30-60 minutes at room temperature or 4 degrees C, protected from light when fluorophores are present.
6
Wash and add secondary if needed
Wash 2 times with permeabilization buffer. If the primary antibody is unconjugated, add fluorophore-conjugated secondary antibody for 20-30 minutes, then wash 2 times.
7
Resuspend and acquire
Resuspend cells in 200-500 uL staining buffer. Acquire samples promptly or store fixed stained cells at 4 degrees C protected from light for a short period if compatible with the fluorophores and assay.
Surface plus intracellular panelsConfirm that surface antibodies tolerate fixation and permeabilization before combining them with intracellular targets.
Cell lossUse low-retention tubes or V-bottom plates, and avoid harsh aspiration when working with limited samples.

Phospho-flow protocol

Use this workflow for phosphorylation or signaling-state analysis by flow cytometry.

  • Use this format when: Measuring pathway activation, inhibitor response, stimulation kinetics, or modification-specific intracellular signal.
  • Optimize first: Stimulation timing, fixation speed, methanol compatibility, antibody titration, and positive or inhibited controls.
1
Prepare stimulation plan
Set up untreated, stimulated, inhibited, and positive control samples. Time stimulation carefully because phosphorylation can change within minutes.
2
Fix immediately
At each time point, fix cells quickly to preserve signaling state. Add pre-warmed fixation buffer to reach the recommended final concentration, commonly 1-4 percent paraformaldehyde, and incubate 10-15 minutes.
3
Permeabilize
Wash cells, then permeabilize using cold 90 percent methanol added slowly while vortexing gently, or use a phospho-flow permeabilization kit. Incubate on ice or at -20 degrees C for 10-30 minutes depending on the protocol.
4
Rehydrate and block
Wash cells 2 times with staining buffer to remove methanol. Block in staining buffer with 1-5 percent BSA or serum for 10-20 minutes.
5
Add phospho/PTM antibody
Dilute NDUFS4 Recombinant Monoclonal Antibody [9H1] in staining buffer. Use the datasheet-recommended flow dilution or titrate across a range. Incubate 30-60 minutes at room temperature or 4 degrees C.
6
Wash and detect
Wash 2 times. Add fluorescent secondary antibody if needed and incubate 20-30 minutes protected from light. Wash again and resuspend for acquisition.
7
Acquire and analyze
Use unstimulated and stimulated controls to set gates. Compare median fluorescence intensity rather than percent positive when signal shifts are continuous.
Methanol sensitivitySome surface fluorophores and epitopes do not tolerate methanol. Stain methanol-sensitive surface markers after permeabilization only if compatible, or choose resistant clones/fluorophores.
KineticsRun pilot time courses when measuring signaling events.

Multicolor flow cytometry panel protocol

Use this workflow to combine multiple antibody markers in one flow cytometry panel.

  • Use this format when: Multiple cell populations or markers need to be measured in the same sample.
  • Optimize first: Fluorophore brightness, marker density, spillover, antibody titration, compensation controls, and fluorescence minus one controls.
1
Design panel
Assign bright fluorophores to low-abundance markers and dimmer fluorophores to high-abundance markers. Avoid placing highly co-expressed markers in channels with severe spillover.
2
Titrate each antibody
Titrate each antibody individually using the same cell type and staining conditions planned for the experiment. Choose the lowest antibody amount that gives clear separation with acceptable background.
3
Prepare cells and Fc block
Aliquot 0.5-1 x 10^6 cells per sample. Wash and block Fc receptors for 10-15 minutes at 4 degrees C.
4
Add antibody cocktail
Prepare a master mix containing all antibodies at final optimized concentrations. Include NDUFS4 Recombinant Monoclonal Antibody [9H1] if it is part of the panel. Add the cocktail to cells in 50-100 uL final staining volume and incubate 20-30 minutes at 4 degrees C protected from light.
5
Wash and fix if needed
Wash 2 times with staining buffer. Fix cells if required by biosafety policy or if acquisition will be delayed. Confirm that fluorophores tolerate fixation.
6
Prepare controls
Prepare unstained cells, single-color compensation controls, viability-only control, and fluorescence minus one controls for critical gates. Include biological positive and negative controls when available.
7
Acquire and gate
Acquire enough events for rare populations. Use compensation controls to correct spillover and FMO controls to place gates for dim or continuous markers.
Panel complexityA good 4-color panel with strong controls is more useful than an overloaded panel with poor compensation.
Viability dyeInclude a viability dye when dead cells could increase nonspecific antibody binding.
Open full FC protocol

Choose Your ELISA Format

Use this ELISA protocol guide to choose and run a plate-based immunoassay workflow. Select sandwich, indirect, direct, or competitive ELISA to view detailed benchtop starting conditions, including coating, blocking, washing, antibody incubation, detection, plate reading, controls, and troubleshooting.

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 low-abundance soluble antigens in complex biological matrices where a compatible matched antibody pair is available.
  • Optimize first: Capture antibody concentration, detection antibody concentration, sample dilution, blocking buffer, wash stringency, standard curve range, and matrix effects.
Sample types
  • Best sample types: Serum, plasma, saliva, culture supernatants, and tissue homogenates.
  • Best for: Quantifying low-abundance antigens, such as cytokines, hormones, and biomarkers, in complex biological matrices.
  • Accuracy note: Sandwich ELISA is usually not the best choice for very small or single-epitope analytes because two non-overlapping antibody binding sites are needed.
1
Prepare plate map and reagents
Plan 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 antibody
Dilute 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 block
Wash 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 samples
Prepare standards in duplicate or triplicate using the recommended diluent. Dilute samples so readings fall within the linear range. For cell lysates or tissue homogenates, prepare clarified samples and include fresh protease inhibitor during upstream extraction when compatible with the assay, such as R-1101 Protease Inhibitor Cocktail. 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 antibody
Dilute detection antibody in assay diluent. If this product is used as the detection antibody, add NDUFS4 Recombinant Monoclonal Antibody [9H1] 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 substrate
Add 100 uL per well HRP-conjugated secondary antibody, or R-1098 Streptavidin: HRP Conjugate when the detection antibody or binding reagent is biotin-labeled. 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 read
Develop 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.

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, purified antigen, semi-purified antigen, or target-specific direct detection reagent is available.
  • Optimize first: Coating or sample input, target purity, detection reagent amount, incubation time, wash stringency, and substrate development time.
Sample types
  • Best sample types: Purified or semi-purified antigens, recombinant proteins, and clarified cell or tissue lysates when target coating and background are acceptable.
  • Best for: Screening antibody specificity or confirming the presence of an antigen in an isolated, high-purity state.
  • Accuracy note: Direct ELISA is simple and fast, but it is less ideal for crude complex matrices because non-target proteins can compete for plate binding and increase background.
1
Prepare plate and samples
Plan standards, blanks, negative controls, positive controls, and sample dilutions. For manual direct ELISA, use purified or semi-purified antigen, recombinant protein, or clarified lysate only when plate coating and background are acceptable. Add 100 uL per well of standards or samples according to the kit format or coating requirement.
2
Immobilize target if required
For 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 block
Wash 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 reagent
Add 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 develop
Wash 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 read
Add 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.

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: Measuring antibody binding, total antibody concentration, titer, or immune response to a coated antigen.
  • Optimize first: Antigen coating concentration, sample or antibody dilution, blocking buffer, secondary antibody dilution, and wash stringency.
Sample types
  • Best sample types: Serum, plasma, saliva, and other body fluids.
  • Best for: Measuring total antibody concentrations, titers, or screening immune responses against specific pathogens or antigens.
  • Accuracy note: Match the secondary antibody to the sample antibody species and isotype, and validate saliva or other non-serum matrices before relying on quantitative comparisons.
1
Coat antigen or target
Dilute 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. If the antigen or capture reagent is biotinylated, a streptavidin-coated plate such as R-1102 Streptavidin-Coated Strip Microwell Plate may be used instead of passive coating. Incubate overnight at 4 degrees C or 2 hours at room temperature.
2
Wash and block
Aspirate 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 antibody
Dilute NDUFS4 Recombinant Monoclonal Antibody [9H1] 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 antibody
Wash 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 antibody
Dilute HRP- or AP-conjugated secondary antibody in assay diluent, commonly 1:2000-1:10000 depending on supplier and signal strength. For common mouse or rabbit primary antibodies, host-matched options include A12003 HRP-Goat Anti-Mouse Secondary Antibody or A12004 HRP-Goat Anti-Rabbit Secondary Antibody. Add 100 uL per well and incubate 30-60 minutes at room temperature.
6
Develop signal
Wash 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 results
Subtract 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.

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, standard curve range, and matrix interference.
Sample types
  • Best sample types: Serum, plasma, saliva, culture supernatants, and crude biological fluids.
  • Best for: Detecting small molecules, hormones, or targets with only a single antigenic epitope.
  • Accuracy note: Competitive ELISA produces an inverse signal, so higher analyte levels usually give lower absorbance or fluorescence.
1
Prepare standards and samples
Prepare a full standard curve using the assay diluent recommended for the target. For serum, plasma, saliva, culture supernatants, or crude biological fluids, 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 competitor
Add 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 reagent
Add the antibody or target-specific binding reagent at the optimized concentration. When using an antibody-based format, add NDUFS4 Recombinant Monoclonal Antibody [9H1] only if the product is the intended competitive binding antibody and follow the datasheet dilution first.
4
Incubate competition reaction
Incubate 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 thoroughly
Wash 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 read
Add 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.
Open full ELISA protocol

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