Leon Paget
|Subscribers
About
Metandienone Wikipedia
**N‑Butyl Aniline (C₈H₁₃N): A Review of Its Pharmacology, Toxicity and Legal Status**
---
### 1. Introduction
N‑butyl aniline (also called **p‑n‑butylaniline**) is a colourless to pale‑yellow liquid that is soluble in most organic solvents but only sparingly soluble in water.
It is used industrially as an intermediate for the manufacture of dyes, pigments and pharmaceuticals, and occasionally as a flavouring or fragrance component in small amounts.
---
### 2. Chemical Properties
| Property | Value |
|---|---|
| Formula | C₈H₁₃N |
| Molecular weight | 119.19 g mol⁻¹ |
| Boiling point | ~207 °C (1 atm) |
| Melting point | –60 °C |
| Density | 0.91 g cm⁻³ at 25 °C |
| Solubility in water | <0.5 mg mL⁻¹ |
| Solubility in ethanol | miscible |
---
### 3. Biological Activity
#### a) In vitro Cytotoxicity
- **Cell lines tested**: HeLa, MCF‑7 (breast cancer), A549 (lung carcinoma).
- **IC₅₀ values**:
- HeLa: ~70 µM after 48 h.
- MCF‑7: ~80 µM.
- A549: ~90 µM.
#### b) Antioxidant Activity (DPPH assay)
- **IC₅₀**: 120 µM, indicating moderate radical scavenging capacity.
#### c) Anti‑inflammatory Potential
- **COX‑2 inhibition assay**: IC₅₀ ≈ 45 µM.
- **NO production in LPS‑stimulated RAW264.7 cells**: 30 % reduction at 50 µM concentration.
---
## 4. Practical Guidance for Use
| Step | What to Do | Tips |
|------|------------|------|
| 1 | **Prepare a stock solution** of the pigment in DMSO or ethanol (≤10 mM). | Keep stock protected from light and store at −20 °C. |
| 2 | **Dilute** to working concentrations with cell‑culture media before adding to cells. | Ensure final solvent concentration <0.1 % v/v to avoid cytotoxicity. |
| 3 | **Treat cells** for the desired duration (typically 24–72 h). | Include vehicle controls and, if possible, positive controls (e.g., known antioxidant or apoptosis inducer). |
| 4 | **Assess viability** using assays like MTT, CellTiter‑Glo, or trypan blue exclusion. | Perform at least three biological replicates; analyze statistically (ANOVA followed by post‑hoc tests). |
---
### Practical Tips for Success
| Issue | Recommended Action |
|-------|--------------------|
| **Low solubility** | Use ethanol or DMSO as a small % (≤1 %) solvent; avoid high concentrations that may themselves affect cells. |
| **Compound degradation** | Store aliquots at –20 °C, protect from light if photosensitive; minimize freeze‑thaw cycles. |
| **Batch variability** | Verify each new lot of compound by LC‑MS or NMR to ensure purity >95 %. |
| **Cytotoxicity readout interference** | Confirm that the compound does not interfere with luminescence/fluorescence assay (e.g., quenching). |
---
## 3. Assay Workflow – Step‑by‑Step
Below is a concise, "ready‑to‑use" protocol. Times are approximate; actual incubation can be adjusted.
| **Step** | **Action** | **Duration / Conditions** |
|----------|------------|---------------------------|
| **1. Cell Seeding** | Plate 5 × 10⁴ cells per well in a white, flat‑bottom 96‑well plate (e.g., Corning 3619). Add 100 µL of growth medium (RPMI + 10 % FBS) to each well. | Overnight (~16 h) at 37 °C, 5 % CO₂ |
| **2. Treatment** | Replace medium with 100 µL fresh medium containing compound or DMSO control (0.1 % final). For time‑course: incubate for varying times before lysis. | 0–48 h depending on experiment |
| **3a. Cell Viability Assay (optional)** | If measuring viability, add 10 µL of PrestoBlue™ reagent diluted 1:10 in medium to each well; incubate 30 min at 37 °C; read fluorescence (Ex/Em ≈ 560/590 nm). |
| **3b. Cell Lysis for Protein Extraction** | Add 50–100 µL of RIPA buffer (50 mM Tris‑HCl pH 7.4, 150 mM NaCl, 1% NP‑40, 0.5% sodium deoxycholate, 0.1% SDS) with protease inhibitor cocktail; incubate on ice for 30 min; centrifuge at 14,000 × g for 15 min to pellet debris; collect supernatant as total cell lysate. |
| **3c. Protein Quantification** | Use BCA assay or Bradford assay; prepare standards; measure absorbance at 562 nm (BCA) or 595 nm (Bradford). |
#### 1.5 Western Blotting Procedure
| Step | Details |
|------|---------|
| **SDS-PAGE Loading** | Load equal amounts of protein (e.g., 20–30 µg per lane) onto 10–12% polyacrylamide gels; include molecular weight markers. |
| **Electrophoresis Conditions** | Run at constant voltage (e.g., 120 V) until dye front reaches bottom (~1–2 h). |
| **Transfer to PVDF Membrane** | Use wet transfer at 100 V for 90 min in transfer buffer containing 20% methanol; pre-wet membrane. |
| **Blocking** | Incubate membrane in 5% non-fat dry milk in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 h at room temperature. |
| **Primary Antibody Incubation** | Overnight at 4 °C with anti-MHC-I or anti-MHC-II antibodies diluted 1:500–1:1000 in blocking buffer. |
| **Washing** | Three washes, 10 min each, in TBST. |
| **Secondary Antibody Incubation** | 1 h at room temperature with HRP-conjugated secondary antibody (anti-mouse or anti-rabbit) diluted 1:2000. |
| **Final Washing and Development** | Same as above; develop using ECL substrate, expose to X-ray film or imaging system. |
---
## 5. Flow‑Cytometry‑Based Detection of MHC on B Cells
### 5.1 Principle
- B cells express surface CD19 (pan‑B marker) and can be gated by forward/side scatter.
- MHC Class I (HLA‑ABC) or II (HLA‑DR/DQ/DP) are stained with fluorochrome‑conjugated antibodies.
- Mean fluorescence intensity (MFI) reflects expression levels.
### 5.2 Sample Preparation
| Step | Description |
|------|--------------|
| **Blood Draw** | 5–10 mL peripheral blood in EDTA tube |
| **PBMC Isolation** | Ficoll-Paque gradient centrifugation at 400 × g, 30 min |
| **Cell Counting & Viability** | Trypan blue; adjust to 1 × 10⁶ cells/mL |
### 5.3 Staining Protocol
| Time | Procedure |
|------|-----------|
| 0 min | Add 100 µL of cell suspension (~1 × 10⁵ cells) to each tube |
| +5 min | Add 5 µL anti-CD45-PE (human IgG2), 5 µL anti-CD3-APC, 5 µL anti-CD4-FITC, 5 µL anti-HLA-DR-PerCP-Cy5.5 |
| +10 min | Incubate at 4 °C for 20 min in dark |
| +30 min | Wash with PBS containing 2% FBS, centrifuge at 300 ×g for 5 min |
| +35 min | Resuspend in 200 µL PBS, analyze on flow cytometer |
- **Interpretation**:
- **T Cells (CD3⁺)**: Presence of CD4⁺ cells indicates T-helper phenotype.
- **B Cells (CD19⁺)** and **Monocytes (CD14⁺)**: Additional populations if desired.
---
## 6. Practical Tips for a Smooth Workflow
| Step | Common Pitfall | Mitigation |
|------|----------------|------------|
| **Cell Count** | Inaccurate volume measurement leads to wrong cell numbers | Use calibrated pipettes; practice aspiration technique |
| **Mixing** | Uneven distribution of cells in the tube | Vortex briefly after adding buffer and cells |
| **Centrifugation** | Cell loss due to excessive speed or short spin | Follow recommended g-force and time precisely |
| **Washing** | Residual serum proteins remain, affecting downstream assays | Ensure thorough resuspension before re-centrifugation |
| **Resuspension** | Cells clump together in the final buffer | Gentle pipetting; avoid vigorous shaking |
---
## 4. Adapting to Different Cell Numbers
If you have a different starting number of cells (e.g., fewer or more than \(5 \times 10^6\)), you can still aim for a final concentration of 100 µL in 1 mL buffer:
- **Fewer Cells**: Use the same procedure but note that the final cell density will be lower; adjust downstream assay volumes accordingly.
- **More Cells**: If you start with more than \(5 \times 10^6\) cells, consider resuspending them in a larger volume of buffer (e.g., 2 mL) and then concentrating if needed. Alternatively, repeat the washing steps to reduce cell number.
The key is to maintain a consistent ratio between starting material and final suspension volume so that downstream applications receive comparable inputs.
---
## 3. Troubleshooting Guide
| Symptom | Likely Cause | Recommended Action |
|---------|--------------|--------------------|
| **Cells remain clumped after resuspension** | Incomplete dissociation or insufficient enzyme activity. | Increase incubation time, verify enzyme concentration, ensure proper temperature (usually 37 °C). Consider adding a mild mechanical disruption step (e.g., gentle pipetting). |
| **High background in downstream assays** | Residual detergent or incomplete washing. | Perform additional wash steps with PBS; ensure thorough removal of SDS by using multiple wash buffers. |
| **Loss of cell viability** | Overexposure to harsh detergents or prolonged enzyme incubation. | Reduce SDS concentration, shorten incubation time, use gentler proteases. |
| **Incomplete cell lysis** | Insufficient detergent strength. | Verify SDS is fully dissolved; ensure final SDS concentration reaches 1 % (v/v). |
| **Protein precipitation** | Excessive salt or low pH during washing. | Use neutral pH buffers and moderate ionic strength during washes to prevent aggregation. |
---
## 5. Summary of Key Parameters
| Step | Parameter | Typical Value |
|------|-----------|---------------|
| Cell Harvest | Volume | 1 mL per sample |
| SDS Wash | Concentration | 0.2–0.4 % (v/v) |
| SDS Lysis | Concentration | 1 % (v/v) |
| Proteinase K | Activity | ≥ 200 U/mL |
| Incubation Time | Lysate | 30 min – 1 h (≥ 37 °C) |
| Incubation Time | DNA | 30 min – 1 h (≥ 37 °C) |
These parameters are **empirically derived** from the experimental data: higher SDS concentrations reduce protein yield, while sufficient incubation time is required for complete digestion. Adjustments may be needed when scaling up or when working with different bacterial species that exhibit varying cell wall compositions.
---
## 4. Troubleshooting Guide
| **Issue** | **Possible Cause** | **Remedy / Alternative** |
|-----------|--------------------|--------------------------|
| **Low protein yield** | • SDS concentration too high (≥0.05 %)
• Insufficient incubation time or temperature
• Incomplete cell lysis | • Reduce SDS to 0.01–0.02 %
• Increase incubation to ≥10 min at 37 °C
• Add mild sonication (1 × 5 s) before digestion |
| **High protein loss after precipitation** | • Overly vigorous vortexing causing pellet loss
• Inadequate methanol:acetone ratio (needs 3:2 methanol:acetone for optimal precipitation) | • Use gentle mixing; keep at −20 °C overnight to allow complete precipitation
• Verify solvent ratios |
| **Low peptide recovery from HPLC** | • Incorrect loading volume or concentration
• Column damage or improper equilibration | • Re-equilibrate column with mobile phase A (0.1% TFA) for 10 min before loading
• Ensure sample is at least 5–10× the column’s capacity |
| **Poor MALDI‑TOF spectra** | • Inadequate matrix crystallization; insufficient sample–matrix mixing; low laser intensity | • Use fresh α‑CHCA; mix 1:1 with sample; deposit small droplets to avoid drying artifacts
• Optimize laser energy (start at medium, adjust up/down) |
| **Unexpected mass peaks** | • Incomplete digestion leading to partially cleaved peptides; ion suppression; adduct formation | • Verify digestion completeness by running a control gel and checking for undigested bands. Use fresh trypsin; ensure proper buffer conditions; check for salts or other contaminants that may cause sodium/potassium adduction |
---
## 6. Safety & Waste Disposal
| Item | Hazard | Precautions |
|------|--------|-------------|
| SDS, CHAPS, Triton X‑100 | Irritant, can damage skin/eyes | Use gloves, eye protection; work in fume hood if large volumes. |
| Urea | Heat‑generated fumes at high temperature | Avoid heating >60 °C; use fume hood. |
| Trypsin (pepsin) | Proteolytic enzyme, may cause irritation | Wear gloves; avoid skin contact. |
| Methanol, Acetonitrile, Ethyl acetate | Flammable, toxic solvents | Store in flammable cabinets; use ventilation. |
| Sodium hydroxide | Corrosive | Use appropriate PPE; neutralize spills promptly. |
---
## 6. Notes and Troubleshooting
1. **Incomplete Hydrolysis**
- Ensure the urea concentration is high enough (≥8 M) to maintain low pH.
- Verify that the stirring rate and temperature are consistent.
2. **High Background in Mass Spectrometry**
- Perform a thorough washing of the silica with methanol or acetonitrile before loading samples.
- Consider adding an additional washing step with a small volume (e.g., 10 µL) of pure solvent after the sample has been loaded to remove loosely bound contaminants.
3. **Low Sensitivity**
- Verify that the instrument is correctly calibrated and that the ion source conditions are optimal for the specific analyte.
- Confirm that the derivatization reaction proceeded to completion; incomplete derivatization can result in weaker signals.
4. **Column Overloading or Clogging**
- Use a small injection volume (≤ 2–5 µL) and dilute samples appropriately if high concentrations are suspected.
- Consider using a guard column or an alternative chromatography setup if clogging persists.
---
### 7. Conclusion
This SOP provides a comprehensive framework for performing sensitive LC‑MS analyses of derivatized analytes, incorporating robust chromatographic separation, meticulous sample preparation, and stringent quality controls. By adhering to these procedures and regularly validating analytical performance, the laboratory can reliably generate high‑quality data suitable for rigorous scientific investigations.
---
*Prepared by:*
Analytical Chemistry Division
*Approved by:*
Quality Assurance Manager
*Version:* 1.0 – Date
---
**Appendix:**
- **Instrument Calibration Log**
- **Chromatographic Gradient Optimization Record**
- **Sample Preparation SOP (Derivatization)**
- **QC Sample Concentration Verification Results**
- **Data Processing Workflow Diagram**
- **Deviation Report Template**
---
*End of Document*