1141251
Descripción
Mapa Mental por Kristi Brogden, actualizado hace más de 1 año
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Creado por Kristi Brogden
hace más de 10 años
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Protein analysis (and interactions)
- Protein separation and identification
- Centrifugation
- Chromatography
- Affinity chromatography
- A wide variety of
ligand coated beads,
from small molecules
to large proteins
- The protein mixture is applied to the
column, with all but the protein with a
specific interaction for the ligand being
washed away in the ‘flow through’.
- Specifically bound proteins
are then eluted using a
competing ligand to dislodge
the affinity interaction.
- Specifically bound proteins
are then eluted using a
competing ligand to dislodge
the affinity interaction.
- The protein mixture is applied to the
column, with all but the protein with a
specific interaction for the ligand being
washed away in the ‘flow through’.
- A wide variety of
ligand coated beads,
from small molecules
to large proteins
- Ion-exchange chromatography
- Increasing salt concentrations are used
to compete for the ionic interactions
between the proteins and the beads. The
proteins are then eluted from the column
- e.g. Cation exchange
- CM carboxy-methyl
Anion exchange
- DEAE diethylaminoethyl
- CM carboxy-methyl
Anion exchange
- Increasing salt concentrations are used
to compete for the ionic interactions
between the proteins and the beads. The
proteins are then eluted from the column
- Affinity chromatography
- SDS-PAGE
- Acrylamide gel concentration has a sieving effect,
and protein migration through the gel in the presence
of SDS is proportional to molecular mass.
- Acrylamide gel concentration has a sieving effect,
and protein migration through the gel in the presence
of SDS is proportional to molecular mass.
- 2D gels
- 1st dimension isoelectric
focussing separates by charge
- 2nd dimension SDS-PAGE
separates based on size
- 2nd dimension SDS-PAGE
separates based on size
- 2-D gels are like a
‘fingerprint’ of the
original protein sample
- Used in proteomics
- 1st dimension isoelectric
focussing separates by charge
- Western blotting
- The gel is electrophoretically
transferred to a membrane.
- Membrane incubated with specific antibody
against component of mixture.
- The ‘primary’ antibody is detected
with a ‘secondary’ antibody with an
enzyme linked to it.
- Western blot ‘developed’ using chemicals that the
linked enzyme converts to colour or light.
- Reaction revealed directly as coloured spots or using a camera.
- Reaction revealed directly as coloured spots or using a camera.
- Western blot ‘developed’ using chemicals that the
linked enzyme converts to colour or light.
- The ‘primary’ antibody is detected
with a ‘secondary’ antibody with an
enzyme linked to it.
- Membrane incubated with specific antibody
against component of mixture.
- Once the position of a protein spot of interest is
identified, it can be cut out of the gel and subjected
to further analysis by mass spectrometry.
- This can be used to determine the
identity of unknown spots, or to identify
post-translational modification of
proteins
- This can be used to determine the
identity of unknown spots, or to identify
post-translational modification of
proteins
- The gel is electrophoretically
transferred to a membrane.
- Centrifugation
- Mass spectrometry
- DNA binding proteins recognise specific DNA sequences
- DNA Footprinting
- Mapping of specific protein-DNA binding
sites to identify, for example regulatory
regions, such as promoters.
- Also to test the regulation of
known DNA-binding proteins by
other factors, such as
phosphorylation or the binding of an
enhancer protein etc.
- Mapping of specific protein-DNA binding
sites to identify, for example regulatory
regions, such as promoters.
- Gel mobility shift assays -
identifying DNA binding proteins
- Affinity purification of DNA binding proteins
- DNA Footprinting
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