MBIO 3410 lecture Nov 20 2007

MBIO 3410 lecture Nov 20 2007 - Last Lecture Overview...

Info iconThis preview shows pages 1–6. Sign up to view the full content.

View Full Document Right Arrow Icon
1 Last Lecture Overview Immunoscreening Antibodies Polyclonal antibodies Monoclonal antibodies Continuous epitopes Discontinuous epitopes Immunoscreening of a λ phage library Screening by Function Phage Display Southern Blotting Detection Southern Blot Stringency Washes High stringency Low stringency
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
2 Northern blotting The detection of RNA sequences using probes RNA is separated on an agarose gel and transferred to a membrane without the alkali step of Southern blots Specific RNA molecules are then detected by hybridization using labeled single-stranded DNA or RNA sequences that are complementary to particular RNAs Northern blots give information about the size of the mRNA and can be useful to determine whether a cDNA clone used as a probe is full-length or whether it is one of a family of related (or alternatively processed) transcripts They can identify whether a genomic clone has regions that are transcribed
Background image of page 2
3 Western blotting The detection of specific proteins using antibodies Proteins are separated through a polyacrylamide gel containing the detergent SDS to keep them in an unfolded ( denatured ) state The proteins are transferred from the gel onto a membrane in much the same way as described above for Southern blotting Particular proteins are then detected using antibodies The specific interaction between the antibody and its antigen occurs on the membrane, and the position of the bound antibody is detected This detection works through the binding of one unlabelled antibody (the primary antibody ) to the antigen on the membrane A second labeled antibody (the secondary antibody ) is then used to detect the presence of the first antibody
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
4 This has several advantages; The multivalent nature of antibody binding means that a substantial increase in sensitivity is achieved A single secondary antibody can be used to detect a number of different primary antibodies e.g. Primary monoclonal antibodies are often raised in mice A secondary antibody raised, say, in the rabbit against mouse immunoglobulins will be capable of interacting with a number of different mouse derived primary antibodies
Background image of page 4
5 Polyacrylamide Gels Polyacrylamide gel electrophoresis (PAGE) is a method for separating proteins The pore size of this kind of gel may be varied, by altering the percentage polyacrylamide used to construct the gel for separating molecules of different sizes from 3 – 30% PAGE is also a powerful technique in the analysis of DNA molecules It is able to very effectively separate DNA molecules that differ in size by as little as a
Background image of page 5

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 6
This is the end of the preview. Sign up to access the rest of the document.

Page1 / 21

MBIO 3410 lecture Nov 20 2007 - Last Lecture Overview...

This preview shows document pages 1 - 6. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online