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Recommended Protocols

Optimized standard operating procedures covering protein labeling, click chemistry, qPCR, cell imaging and routine assays.

AllProtein labelingClick chemistryqPCRCell imagingBasic assays

NHS Ester Dye Protein Labeling

Protein labeling

Standard procedure for covalent labeling of lysine residues on proteins (antibodies, BSA, etc.) using NHS ester-activated fluorescent dyes.

Procedure

  1. 1Dissolve the target protein in 0.1 M NaHCO₃ buffer (pH 8.3–8.5) at 1–10 mg/mL
  2. 2Dissolve the NHS ester dye in anhydrous DMSO or DMF to ~10 mM
  3. 3Add dye solution dropwise with stirring at a dye:protein molar ratio of 10:1 – 20:1
  4. 4React at room temperature, protected from light, for 1–2 h (or overnight at 4°C)
  5. 5Remove unreacted dye using a Sephadex G-25 or PD-10 desalting column
  6. 6Determine the degree of labeling (DOL) from A₂₈₀ and dye λmax absorbances
  7. 7Store the labeled product at 4°C protected from light; addition of 0.02% NaN₃ is recommended

💡 Notes: The buffer must not contain primary amines (Tris, glycine, etc.) as they compete with the labeling reaction. Use carbonate or phosphate buffers.

Maleimide Thiol Labeling

Protein labeling

Site-specific labeling via the selective reaction of maleimide groups with free thiols (cysteine residues) on proteins.

Procedure

  1. 1Dissolve the protein in 0.1 M sodium phosphate buffer (pH 7.0–7.5) containing 5 mM EDTA
  2. 2If the protein lacks free thiols, reduce disulfide bonds with TCEP or DTT (2–10 mM, RT 30 min)
  3. 3Immediately desalt to remove excess reducing agent
  4. 4Add maleimide dye (dissolved in DMSO) at a dye:protein molar ratio of 5:1 – 15:1
  5. 5React at room temperature, protected from light, for 2 h (or overnight at 4°C)
  6. 6Quench unreacted dye with N-ethylmaleimide (NEM, final concentration 1 mM)
  7. 7Desalt and purify; determine the degree of labeling

💡 Notes: Maintain pH 7.0–7.5 throughout — above pH 8, maleimide can react non-specifically with amine groups.

Copper-Catalyzed Click Chemistry (CuAAC)

Click chemistry

Fluorescent labeling of alkyne- or azide-modified biomolecules via azide–alkyne cycloaddition (CuAAC).

Procedure

  1. 1Dissolve the alkyne/azide-modified biomolecule in PBS or Tris buffer
  2. 2Add CuSO₄ solution (0.1–0.5 mM final) and THPTA ligand (Cu:THPTA = 1:5 – 1:10)
  3. 3Add freshly prepared sodium ascorbate (1–5 mM final) as the reducing agent
  4. 4Add the azide/alkyne-modified fluorescent dye (0.1–1 mM final)
  5. 5React at room temperature or 37°C for 30 min – 2 h
  6. 6Remove copper catalyst and unreacted dye by desalting or dialysis

💡 Notes: Optimize copper concentration based on substrate amount. For intracellular labeling, copper-free click chemistry (SPAAC) is recommended.

SYBR Green I qPCR Reaction Setup

qPCR

Standard real-time fluorescent quantitative PCR protocol using Qihe ProMaster® qPCR Mix (containing SYBR Green I).

Procedure

  1. 1Prepare reaction mix on ice (20 μL): 2× ProMaster Mix 10 μL + forward/reverse primers 0.4 μL each (0.2 μM final) + template DNA <100 ng + ddH₂O to 20 μL
  2. 2Mix gently and centrifuge briefly to eliminate bubbles
  3. 3Run on instrument: 95°C 3 min → [95°C 10 s → 60°C 30 s (data acquisition)] × 40 cycles
  4. 4Melt-curve analysis: 95°C 15 s → 60°C 1 min → ramp to 95°C at 0.5°C/s (continuous acquisition)
  5. 5Quantify using the ΔΔCt method or a standard curve

💡 Notes: Three technical replicates are recommended. Primer efficiency within 90–110% is considered acceptable.

Fluorescence In Situ Hybridization (FISH)

Cell imaging

In situ detection of specific nucleic acid sequences in fixed cells/tissue using fluorescently labeled oligonucleotide probes.

Procedure

  1. 1Fix cells or tissue sections (4% PFA, RT 15–20 min); wash 3× with PBS
  2. 2Permeabilize: 0.5% Triton X-100 in PBS, RT 10 min
  3. 3Pre-hybridize: add pre-hybridization solution, incubate at 37–42°C for 30–60 min
  4. 4Hybridize: add fluorescently labeled probe (5–50 ng/μL in hybridization buffer), 37–42°C protected from light for 2–16 h
  5. 5Wash: 2–3× with 2× SSC + 0.1% Tween-20 at 37°C, 5–15 min each
  6. 6Counterstain nuclei with DAPI (1 μg/mL, RT 5 min); wash with PBS
  7. 7Mount with anti-fade mounting medium; image by fluorescence microscopy

💡 Notes: Probe length, GC content and hybridization temperature should be optimized for the target sequence. Keep samples protected from light to prevent photobleaching.

BCA Protein Quantification

Basic assays

Determination of protein concentration by the BCA (Bicinchoninic Acid) method, suitable for most protein and peptide samples.

Procedure

  1. 1Prepare BSA standard curve: 0, 25, 50, 100, 200, 500, 1000, 2000 μg/mL
  2. 2Add 25 μL of each standard and unknown sample to a 96-well plate (in duplicate)
  3. 3Prepare BCA working reagent at a ratio of Reagent A : Reagent B = 50 : 1
  4. 4Add 200 μL BCA working reagent to each well; incubate at 37°C for 30 min
  5. 5Cool to room temperature; read absorbance at A₅₆₂ on a plate reader
  6. 6Plot the standard curve (concentration on x-axis, absorbance on y-axis)
  7. 7Calculate sample protein concentration from the standard curve

💡 Notes: The BCA assay is sensitive to reducing agents (DTT, β-ME) and chelating agents (EDTA); keep their concentrations low.

Need more protocols? Contact our technical support team

info@qihe-bio.com

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