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Monday, November 22

  1. page GST pull-down assay edited GST pull-down assay A GST (glutathione-S-transferase) pull-down assay is a technique used to st…

    GST pull-down assay
    A GST (glutathione-S-transferase) pull-down assay is a technique used to study protein-protein interactions in vitro. It involves the addition of a fusion tag for the GST enzyme (tag = 26kDa) at the N-terminus (5' end) of the gene encoding one of the proteins of interest. The GST-tagged protein can then be purified by virtue of its high affinity for glutathione beads, washed of contaminants, and eluted using competing glutathione which binds preferentially to the beads.
    To demonstrate that protein X interacts with protein Y:
    1. Create a GST-X fusion protein
    2. Use glutathione-coated assay beads to purify GST-X
    3. If GST-X binds Y then Y should also be present on the bead (use a Y-specific antibody to verify this)

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    1:15 am
  2. page Pulse-chase edited Pulse-chase Pulse-chase is a technique used to monitor the rate of protein synthesis in vivo, b…

    Pulse-chase
    Pulse-chase is a technique used to monitor the rate of protein synthesis in vivo, by adding a 'pulse' of labelled 35S-methionine for a set time, and then 'chasing' the radio-label out by incubating cells with an excess of unlabelled methionine. Methionine is chosen because it is incorporated universally as the starting amino acid of all proteins. Cell extracts can be isolated at various time points in this process, proteins immunoprecipitated and then SDS-PAGE is used to quantify the 35S label.

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    1:06 am
  3. page Immunocytochemistry edited ... Immunocytochemistry Immunocytochemistry is a technique used to detect proteins within cells; …
    ...
    Immunocytochemistry
    Immunocytochemistry is a technique used to detect proteins within cells; immunohistochemistry is the equivalent process in tissues.
    ...
    insoluble product. This
    This
    technique allows
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    1:04 am
  4. page Immunocytochemistry edited Immunocytochemistry Immunocytochemistry is a technique used to detect proteins within cells; im…

    Immunocytochemistry
    Immunocytochemistry is a technique used to detect proteins within cells; immunohistochemistry is the equivalent process in tissues.
    First the cells are fixed using formaldehyde to preserve structures and cross-link any proteins. Then the cells are permeabilised, to allow antibody access, using a membrane-disrupting detergent such as Triton X-100, memanol or acetone. Antibodies complementary to any proteins of interest are added and then visualised, either directly or indirectly by the addition of an enzyme- or fluorophore-conjugated secondary antibody. If using an enzyme, then it must produce an insoluble product. This technique allows the determination of which cells express a particular protein, and where in the cell the protein is localised; however, it does not allow this study in vivo since the cell has to be killed. Fluorescent protein tagging has become more common in recent years for the study of living interactions between proteins.

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    1:02 am
  5. page Chromatin immunoprecipitation edited ... Chromatin immunoprecipiation Chromatin immunoprecipitation (also chIP) is a technique used to…
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    Chromatin immunoprecipiation
    Chromatin immunoprecipitation (also chIP) is a technique used to determine the particular DNA sequences bound by specific proteins.
    ...
    purify the DNA (theDNA, while the protein is discarded).discarded. The DNA
    1. PCR: using primers against a gene of interest to see if it interacts with the protein
    2. qPCR: to quantify the level of protein binding
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    12:57 am
  6. page Chromatin immunoprecipitation edited ... 1. PCR: using primers against a gene of interest to see if it interacts with the protein 2. q…
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    1. PCR: using primers against a gene of interest to see if it interacts with the protein
    2. qPCR: to quantify the level of protein binding
    3. Microarray analysis:analysis (also chIP-on-chip): to determine
    ...
    relative affinities (i.e. have competing fluorescence between cDNAs from chIP analysis and cDNAs from unbound DNA)
    4. chIP-seq: to determine all the sequences bound by the protein
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    12:56 am
  7. page Chromatin immunoprecipitation edited Chromatin immunoprecipiation ... to determine whether the particular DNA sequences bound by …

    Chromatin immunoprecipiation
    ...
    to determine whetherthe particular DNA sequences bound by specific proteins.
    First the DNA and proteins are cross-linked (fixed together) by heat or UV treatment, then chromatin is isolated and fragmented by either sonication or digestion. Then immunoprecipitation is performed against the protein of interest (whether
    a transcription factor, acetylated histone, etc) and cross-linking is reversed to purify the DNA sequence(the protein is discarded). The DNA is then amplified by PCR for analysis:
    1. PCR: using primers against a gene of interest to see if it interacts with the protein
    2. qPCR: to quantify the level of protein binding
    3. Microarray analysis: to determine if the protein binds to a large number of sequences, as well as relative affinities
    4. chIP-seq: to determine all the sequences
    bound by histone proteins.the protein
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    12:55 am
  8. page Chromatin immunoprecipitation edited Chromatin immunoprecipiation Chromatin immunoprecipitation (also chIP) is a technique used to d…

    Chromatin immunoprecipiation
    Chromatin immunoprecipitation (also chIP) is a technique used to determine whether a DNA sequence is bound by histone proteins.

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    12:49 am
  9. page Immunoprecipitation edited Immunoprecipitation Immunoprecipitation is a method used for protein detection in a sample wher…

    Immunoprecipitation
    Immunoprecipitation is a method used for protein detection in a sample where proteins are expressed at low levels, have been post-translationally modified, or form parts of larger complexes or interactions.
    Antibodies specific to the protein of interest are used to remove (precipitate) that protein from solution by adding the antibody and then adding a molecule complementary to the antibody that is insoluble, before spinning the solution to precipitate the antibody-protein complex and eluting it with SDS.
    When detecting post-translational modifications of a protein, the insoluble antibody-binding protein used should be an anti-GATA-1. Then immunoprecipiated proteins should then be run on a gel, transferred to a membrane by Western blotting, and probed with an antibody against modified (acetylated) GATA-1.
    Co-immunoprecipitation is the method used to identify proteins in complexes. An organelle (e.g. the nucleus) is first isolated by centrifugation and then an antibody is added that is specific to one of the proteins in the complex of interest. Then insoluble antibody-binding beads are added, and the immunoprecipitated proteins are washed and collected. Finally, a Western blot analysis is performed but this time using antibodies specific to the second protein in the complex of interest; if the two proteins interact then immunoprecipitation of one should enable the detection of the other. A 'pre-clearing' step should be performed to remove non-specifically bound protein; Marvel is often used to prevent non-specific protein binding.

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    12:42 am
  10. page Immuno dot blot edited Immuno dot blot An immuno dot blot is a method used to detect a particular protein in a heterog…

    Immuno dot blot
    An immuno dot blot is a method used to detect a particular protein in a heterogeneous protein extract mixture. Protein extracts from various tissues are placed in each of their own spots on a nitrocellulose membrane before being incubated with an antibody specific to the protein of interest. Excess antibody is washed off and the antibody visualised; the spot on the membrane which gives a signal is the tissue expressing the protein of interest.

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    12:32 am

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