Enzyme-Linked Immunosorbent Assay (ELISA)

Key Steps in the ELISA Procedure

Coating the Plate:

 Antigen or Antibody Attachment: The wells of a microplate (usually 96-well) are coated with
the antigen (the target molecule) or an antibody that will capture the target antigen. This is
done by adding the sample and allowing it to incubate, typically at room temperature or
37°C for about 1 hour or overnight at 4°C12.

Blocking:

 After coating, any unbound sites on the plate are blocked using a blocking buffer (like BSA or
casein). This step prevents non-specific binding of other proteins or antibodies to the plate,
which could lead to false positives15.

Adding Detection Antibody:

 A specific enzyme-linked antibody is added, which binds to the target antigen (or to the
primary antibody if using an indirect method). This forms a "sandwich" complex of antigen-
antibody-enzyme124.

Washing:

 The plate is washed multiple times to remove any unbound antibodies. This step is crucial for
ensuring that only bound antibodies remain for detection16.

Substrate Addition:

 A substrate that reacts with the enzyme linked to the detection antibody is added. This
reaction produces a measurable signal, often a color change, which indicates the presence
and quantity of the target antigen25.

Reading Results:

 The intensity of the color change is measured using a spectrophotometer. The amount of
color produced correlates with the concentration of the target antigen in the sample.
Gas Chromatography-Mass Spectrometry (GC-
MS)
Key Steps in the GC-MS Procedure

Sample Injection:

 The sample is injected into the gas

chromatograph (GC) where it is vaporized.
This typically occurs in a heated injector port
at temperatures up to 300°C.

Separation:

 The vaporized sample is carried by an inert gas (commonly helium) through a capillary
column coated with a stationary phase. As the sample travels through the column, its
components are separated based on their size and polarity. Each compound exits the column
at different times, known as retention times.

Ionization:

 The separated compounds enter the mass spectrometer (MS), where they are ionized.
Common methods of ionization include Electron Ionization (EI) and Chemical Ionization (CI).
This process converts neutral molecules into charged ions.

Mass Analysis:

 The ions generated are sorted based on their mass-to-charge ratio (m/z) in the mass
analyzer. This separation allows for the identification of each ion based on its unique mass.

Detection:

 The ions are detected, and their abundance is measured. This data produces a mass
spectrum that indicates the presence and quantity of each compound in the sample.

Data Analysis:

 The resulting mass spectrum is compared against standard reference libraries to identify
unknown substances. Quantification can also be performed based on peak areas in the
chromatogram.
Microscopy
Key Steps in the Microscopy Procedure

Preparation:

 Set Up the Microscope: Uncover the

microscope, plug it in, and ensure all
glass surfaces are clean using lens
paper.
 Light Adjustment: Turn on the light source and adjust the diaphragm to control the amount
of light passing through.

Slide Placement:

 Position the Slide: Place your prepared slide on the stage and secure it using stage clips.
Make sure the specimen is centered over the light source.

Initial Focusing:

 Select Low Power Objective: Start with the lowest power objective lens (usually 4x).
 Coarse Focus: Use the coarse focus knob to bring the stage up towards the objective lens
until you see a blurry image. Then, slowly move it down until the image comes into focus.

Fine Focusing:

 Switch Objectives: Once focused at low power, switch to a higher power objective (10x or
40x) without changing the focus knob.
 Fine Focus Adjustment: Use the fine focus knob to sharpen the image. Center your specimen
again if necessary.

Observation:

 Adjust Lighting: Fine-tune the light intensity and diaphragm for optimal clarity.
 Explore Different Areas: Move the slide to examine different parts of your specimen.

Cleanup:

 Return to Low Power: Switch back to the lowest power objective before removing the slide.
 Clean Up: Turn off the light, clean any spills, and cover the microscope when not in use.
Liquid Chromatography-Mass Spectrometry (LC-
MS)

Key Steps in the LC-MS Procedure

Sample Preparation:

 Dissolve Sample: The sample is dissolved in a

suitable solvent (mobile phase) to create a
solution that can be injected into the LC system.

Injection:

 Inject Sample: The prepared sample solution is injected into the liquid chromatography
system using an autosampler or manual syringe.

Separation:

 Column Separation: The sample travels through a chromatographic column where its
components are separated based on their interactions with the stationary phase and mobile
phase. Different compounds will pass through at different rates, leading to their separation.

Ionization:

 Ionization Source: After separation, the eluted components enter the mass spectrometer,
where they are ionized. Common ionization methods include Electrospray Ionization (ESI)
and Atmospheric Pressure Chemical Ionization (APCI), which convert the molecules into
charged ions.

Mass Analysis:

 Mass Spectrometer: The ionized compounds are directed into a mass analyzer, where they
are separated based on their mass-to-charge ratio (m/z). This allows for precise identification
of each compound.

Detection and Data Analysis:

 Detection: The separated ions are detected, and their abundance is measured. This data
generates a mass spectrum, which plots the m/z against intensity.
 Data Interpretation: The resulting mass spectrum is analyzed to identify and quantify the
compounds present in the sample by comparing it to known standards or libraries.
Atomic Absorption Spectroscopy (AAS)
Key Steps in the AAS Procedure

Sample Preparation:

 Dissolve Sample: Prepare your sample by

dissolving it in a suitable solvent, typically
water or an acid solution, to ensure that
the analyte is in liquid form.

Calibration:

 Prepare Standards: Create a series of standard solutions with known concentrations of the
metal ions you wish to analyze.
 Calibration Curve: Measure the absorbance of each standard using the AAS instrument to
generate a calibration curve, which will be used to determine the concentration of metals in
your samples.

Instrument Setup:

 Turn On Equipment: Start the AAS instrument and allow it to warm up for about 20 minutes
to stabilize the light source.
 Align Burner and Set Parameters: Align the burner and configure the software settings
according to the specific metal being analyzed.

Sample Injection:

 Introduce Sample: Use a sipper tube to draw your sample solution into the instrument.
Ensure that the sipper tube remains immersed in a solution between readings to prevent
contamination.

Measurement:

 Analyze Samples: The instrument measures the absorbance of light at specific wavelengths
corresponding to the metal ions present in your sample. The amount of light absorbed is
proportional to the concentration of the metal in the sample.

Data Analysis:

 Interpret Results: Compare the absorbance readings from your samples against the
calibration curve to determine their concentrations.

Cleanup:

 Rinse System: After completing measurements, rinse the sipper tube with distilled water or a
blank solution to clean it.
 Shutdown Procedure: Turn off the flame and spectrometer, and allow any components that
were heated to cool down before cleaning them with nitric acid if necessary.
Inductively Coupled Plasma Mass
Spectrometry (ICP-MS):
Key Steps in the ICP-MS Procedure

Sample Preparation:

 Dissolve Solid Samples: If analyzing solid

samples, they must be digested in
strong acids (like nitric acid) to convert
them into a liquid form. For liquid
samples, ensure they are adequately
diluted, often using a matrix with low total dissolved solids (TDS), typically below 0.2% to
prevent clogging the nebulizer.

Sample Introduction:

 Nebulization: The prepared liquid sample is introduced into the ICP-MS using a nebulizer,
which converts the liquid into a fine aerosol mist. This mist is carried into the plasma by an
argon gas stream.

Ionization:

 Plasma Generation: The aerosol enters an inductively coupled plasma torch, where it is
subjected to high temperatures (around 10,000 K). This causes the sample to desolvate,
atomize, and ionize, generating charged ions from the sample elements.

Ion Extraction:

 Interface Region: The ions produced in the plasma are extracted through a series of cones
(interface cones) that help filter out neutral particles and reduce background noise.

Mass Analysis:

 Mass Spectrometer: The extracted ions are directed into a mass analyzer (often a
quadrupole), which separates the ions based on their mass-to-charge ratio (m/z). This allows
for the identification of different elements present in the sample.

Detection and Data Analysis:

 Ion Detection: The separated ions are detected by a detector (like an electron multiplier),
which counts the number of ions per second and produces a mass spectrum.
 Quantification: The concentration of each element is determined by comparing the ion
counts with those from calibration standards.

Cleanup:

 After analysis, thoroughly rinse any sample introduction components to prevent

contamination for future analyses.
Capillary Electrophoresis (CE)
Key Steps in the CE Procedure

Sample Preparation:

 Dissolve Sample: Prepare your

sample by dissolving it in an
appropriate buffer solution.
Ensure that the sample
concentration is suitable for
analysis, typically between 0.1 to
10 mg/mL.

Capillary Conditioning:

 Condition the Capillary: Rinse the capillary with a conditioning solution (often a strong acid
or base) to remove any contaminants and ensure proper separation conditions. This step
may also include rinsing with the running buffer.

Loading the Sample:

 Inject Sample: Introduce the prepared sample into the capillary. This can be done using
pressure or vacuum injection methods, depending on the instrument setup.

Electrophoresis:

 Apply Voltage: Connect the capillary to a high-voltage power supply and apply an electric
field. The ions in the sample will migrate through the capillary based on their charge and
size, with smaller and more highly charged ions moving faster.

Detection:

 Monitor Separation: As the ions separate, they pass through a detector (such as UV-Vis or
fluorescence), which measures their concentration at specific times. The output is typically
displayed as an electropherogram, showing peaks corresponding to different components.

Data Analysis:

 Analyze Results: Review the electropherogram to identify and quantify the separated
components based on their migration times and peak areas.

Cleanup:

 Rinse Capillary: After analysis, rinse the capillary with buffer or cleaning solutions to prepare
it for future runs.