Technical Support
by Application Area

Confirm the target is expressed in the sample and that the antibody is validated for your application and species. Check storage history, dilution, incubation time and detection reagents. Run a known-positive control and titrate the primary antibody around the datasheet recommendation.

Reduce primary or secondary concentration, improve blocking, extend washes and verify that the secondary antibody matches the primary host. For phospho-targets, use BSA rather than milk. Include no-primary and isotype controls where appropriate.

Start with the datasheet range, then test a small dilution series around it using the same sample load, incubation time and detection settings. Include a known-positive sample and a no-primary control. Choose the lowest antibody concentration that preserves specific signal without unacceptable background.

Confirm that the target sequence or epitope is conserved in the sample species and that the primary antibody is validated for that species and application. The secondary antibody must recognise the primary antibody host species and immunoglobulin class, and should be cross-adsorbed when closely related sample species may cause background.

Compare the new and current lots side by side using the same positive and negative controls, sample preparation, dilution, incubation and instrument settings. Record signal, background and localisation or band pattern before changing routine protocols.

Follow the product datasheet for buffer and concentration. Add buffer gently, allow the vial to stand, then mix without vigorous vortexing. Prepare single-use aliquots in low-binding tubes and avoid repeated freeze-thaw cycles.

Review storage temperature, buffer composition, concentration and freeze-thaw history. Aggregation may occur after vigorous mixing, prolonged room-temperature exposure or storage near the isoelectric point. Compare a fresh aliquot and use an appropriate positive-control assay.

Use the product datasheet because suitable pH, ionic strength, cofactors, reducing agents and carrier proteins depend on the molecule and assay. If a buffer exchange is required, test a small aliquot first and avoid conditions near the protein's isoelectric point when aggregation is a concern.

For a purified protein, A280 can be used when the sequence-based extinction coefficient and path length are known. A compatible colorimetric assay can be used when excipients interfere with absorbance. Measure against the correct blank and avoid assuming that total protein concentration equals active protein concentration.

Include a reagent blank, vehicle control, known active positive control and a concentration series of the test protein. Keep buffer composition and incubation conditions matched. For enzymes, confirm that substrate conversion is measured within the initial linear range.

The most common causes of no-band results are:

• Primer failure - check Tm, GC content (40-60%), and that primers don't form dimers
• Degraded template - run template on gel to confirm integrity; A260/A280 should be 1.8-2.0
• Inhibitors in template - carry-over of ethanol, phenol, or EDTA can block polymerase
• Incorrect Mg²⁺ concentration - standard is 1.5 mM; titrate 1.0-3.0 mM
• Annealing temperature too high - try reducing by 5°C increments

Non-specific amplification is usually caused by low annealing stringency or mispriming.

• Raise annealing temperature in 2°C steps until non-specific bands disappear
• Use a hot-start polymerase to prevent extension at room temperature
• Reduce template concentration - over-loading drives non-specific products
• Add 5% DMSO or betaine for GC-rich targets

For most qPCR assays, 1-10 ng of cDNA or genomic DNA per 20 µL reaction is optimal. Over-loading (> 100 ng) causes inhibition and artificially high Cq values. Under-loading (< 0.01 ng) increases inter-replicate variability.

Always run a standard curve across 4-5 log dilutions to verify reaction efficiency (90-110% acceptable). Efficiency is calculated as: E = (10^(-1/slope)) - 1.

Check template integrity and purity, reverse-transcription consistency, pipetting, primer specificity and reaction efficiency. Run a standard curve, no-template control and no-reverse-transcriptase control where relevant. Replicate variability often increases close to the assay's limit of quantification.

Early amplification with a product-like melt peak may indicate contamination. Late amplification with a lower melt temperature often suggests primer-dimer formation, but confirm by melt analysis or gel electrophoresis. Replace suspect reagents and keep pre-amplification work physically separate from amplified products.

Prepare standards with calibrated pipettes, mix every dilution thoroughly and run duplicates. Confirm the correct curve model for the kit, subtract blanks as instructed and avoid using readings outside the plate reader’s linear range.

High CV commonly results from inconsistent pipetting, bubbles, incomplete mixing, edge effects or unequal incubation times. Use a multichannel pipette consistently, remove bubbles before reading and add reagents in the same sequence across the plate.

Low signal typically points to one of these causes:

• Reagent expiry or freeze-thaw damage - check lot dates; avoid repeated freeze-thaw of antibodies
• Insufficient incubation time or temperature - ensure plate is at correct temperature and incubation not cut short
• Substrate not activated - TMB substrate should be at room temperature before use
• Sample concentration too low - ensure sample is within the standard curve range; concentrate if necessary

Check wash performance, reagent dilution, incubation time, substrate development time and plate drying. Confirm that the conjugate and substrate were prepared correctly and that the plate reader wavelength is appropriate. Repeat with fresh wash buffer and include blank wells to identify reagent background.

Do not extrapolate beyond the validated standard range. Dilute samples above the range and repeat them, then apply the dilution factor. Samples below the lower limit should be reported according to the kit's validated detection and quantification limits rather than assigned an unsupported concentration.

Mycoplasma is the most prevalent cell culture contaminant and is invisible to the eye or standard bright-field microscopy. Detection methods:

• PCR-based detection - most sensitive; ABMIUM Mycoplasma Detection Kit (Cat. AB-MYC-01) detects > 90 species
• DAPI/Hoechst staining - mycoplasma appear as small fluorescent dots around nuclei

For elimination, BM-Cyclin treatment over two 7-day cycles is effective for most strains. Confirm clearance 2 weeks after treatment.

Review passage number, viability, seeding density, medium formulation, supplement quality and incubator temperature, carbon dioxide and humidity. Confirm cell identity and test for mycoplasma. Compare growth with an authenticated, contamination-free reference culture when available.

Check vessel coating, confluence, dissociation time, medium pH and temperature, and the force used during washes or feeding. Examine cultures for contamination and cell death. Some cell types require matrix coating or gradual adaptation after thawing.

Document the change and review confluence, medium, serum or supplement lot, passage number and handling. Quarantine suspect cultures, test for mycoplasma and verify identity if the change persists. Do not rely on morphology alone to confirm identity or contamination.

Freeze healthy cells at an appropriate density using the recommended cryoprotectant and a controlled cooling rate, then transfer promptly to long-term storage. Thaw rapidly, dilute cryoprotectant as the cell type requires and minimise time at room temperature. Assess viability and recovery after attachment or outgrowth, not only immediately after thawing.

Check the product-specific shipping and stability statement before use. Photograph the parcel, record the arrival temperature and contact support with the catalogue number, lot number and delivery date. Do not discard packaging until the case is reviewed.

Provide catalogue and lot numbers, order reference, storage history, protocol, sample type, controls, instrument settings and representative raw data or images. Include the expected result and the observed result.

Stop before using the kit, retain the packaging and photograph the shipment. Record the product, lot, order number and affected component, then contact support. Do not combine components from different lots unless the instructions explicitly allow it.

Check the product-specific instructions before use. Some salts or concentrated buffers can precipitate during cold storage and may redissolve under specified conditions, while unexpected precipitate can indicate contamination or degradation. Do not heat, filter or discard material unless the instructions permit it.

Performance after the stated expiry date is not assured. Use an in-date product for regulated, diagnostic or decision-critical work. For exploratory research, any use beyond expiry should be qualified against an in-date control and documented, but support specifications may no longer apply.