Primary Antibody Standard Protocols in Immunoassay
Primary antibodies are the cornerstone of most immunoassays. Creative Biolabs provides high-quality primary antibodies validated across multiple applications. Below are recommended protocols and optimization guidelines for standard lab applications, developed based on our internal QC testing and extensive user feedback. These protocols serve as a general guide and may require optimization depending on specific experimental conditions.
Standard Western Blotting Protocol
1. Sample Preparation
- Lyse cells/tissues using RIPA or other lysis buffer with protease/phosphatase inhibitors.
- Quantify protein and load 10-50 µg per well.
- Boil in SDS sample buffer at 95°C for 5 minutes.
2. SDS-PAGE and Transfer
- Run samples on an 8-12% polyacrylamide gel.
- Transfer proteins to PVDF or nitrocellulose membrane (semi-dry or wet transfer).
3. Blocking
- Block in 5% non-fat milk or 5% BSA in TBST for 1 hour at room temperature.
4. Primary Antibody Incubation
- Dilute Creative Biolabs' primary antibody in blocking buffer (typically 1:500 to 1:5000).
- Incubate overnight at 4°C or 1-2 hours at room temperature.
5. Washing & Detection
- Wash 3× with TBST, 5 minutes each.
- Proceed with secondary antibody incubation and chemiluminescent detection.
Immunohistochemistry (IHC) Protocol
1. Tissue Preparation
- Deparaffinize FFPE sections and rehydrate through graded alcohols.
- Perform antigen retrieval (e.g., citrate buffer pH 6.0, 95-100°C for 20 minutes).
2. Blocking Endogenous Enzymes
- Incubate in 3% hydrogen peroxide in PBS for 10 minutes to block peroxidase.
3. Blocking Non-Specific Binding
- Block in 5% normal serum or BSA for 30 minutes at room temperature.
4. Primary Antibody Incubation
- Dilute Creative Biolabs' primary antibody as recommended.
- Incubate for 1-2 hours at room temperature or overnight at 4°C.
5. Detection
- Rinse in PBS, proceed with HRP-conjugated secondary antibody and DAB substrate.
Flow Cytometry (FC) Protocol
1. Cell Preparation
- Harvest and wash cells (1-5 × 10⁶ cells/sample).
- Optional: block Fc receptors using species-specific Fc blocking reagent.
2. Primary Antibody Staining
- Dilute Creative Biolabs' primary antibody in staining buffer (PBS + 1% BSA).
- Incubate for 30 minutes at 4°C in the dark.
3. Washing & Analysis
- Wash 2× with staining buffer.
- Proceed with fluorophore-conjugated secondary antibody if needed.
- Analyze via flow cytometer.
Antibody Dilution Optimization
- Always begin with the dilution recommended on the Creative Biolabs datasheet.
- Perform a dilution series (e.g., 1:200 to 1:5000) to determine optimal signal-to-noise ratio.
- Use positive and negative controls for each assay.
Blocking and Dilution Buffers
Creative Biolabs recommends the following buffers depending on assay type:
| Application | Blocking Buffer | Dilution Buffer |
|---|---|---|
| Western Blot | 5% Milk or BSA in TBST | 1% BSA in TBST |
| IHC | 5% Normal Serum or BSA | PBS + 0.05% Tween-20 |
| Flow Cytometry | 2% BSA or 5% FBS in PBS | PBS + 1% BSA + 0.1% NaN₃ |
Ensure buffers are freshly prepared and filtered if needed.
Antibody Concentration Optimization
- Titrate Creative Biolabs antibodies to minimize background and maximize signal.
- For low-abundance targets, use higher concentrations; for high-abundance targets, lower concentrations reduce non-specific binding.
- Include a no-primary control to assess background from secondary antibodies.
Species Cross-Reactivity Considerations
- Always verify the host species of Creative Biolabs' primary antibody and target antigen species.
- Use isotype controls where appropriate.
- Creative Biolabs antibodies are validated for human, mouse, and rat, with cross-reactivity data available in product datasheets. Contact Creative Biolabs technical support for species not listed.
Storage and Freeze-Thaw Cycles
- Store Creative Biolabs' primary antibodies according to the storage conditions specified on the product labeling, or consult our technical support team for confirmation of appropriate handling.
- Avoid repeated freeze-thaw cycles; aliquot upon first thaw if frequent use is expected.
- For short-term use (≤2 weeks), storage at 4°C is generally acceptable.
For further technical support or to request customized protocol recommendations, contact Creative Biolabs' antibody development team. Our specialists are here to help optimize your experimental success with validated reagents you can trust.
