1 / 13

SDS covers proteins in a net negative charge

SDS covers proteins in a net negative charge Addition of 2-mercaptoethanol reduces disulphide bonds and Boiling is used to further denature proteins. Charged R groups. +. -. -. -. +. +. H. -. +. +. Hydrophobic areas. H. +. -. -. -. -. Before SDS. -. -. -. -. -. -. -.

marlin
Download Presentation

SDS covers proteins in a net negative charge

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. SDS covers proteins in a net negative charge Addition of 2-mercaptoethanol reduces disulphide bonds and Boiling is used to further denature proteins. Charged R groups + - - - + + H - + + Hydrophobic areas H + - - - - Before SDS - - - - - - - - - - - - - Migrate in gel according to mass

  2. Proteins are separated in a ‘discontinuous’ system. Stacking gel has looser pores to allow proteins to line up first. How does an SDS-PAGE gel really work? http://mullinslab.ucsf.edu/Protocols%20HTML/SDS_PAGE_protocol.htm

  3. Western blots- Ab used to identify Ag immobilized on nylon

  4. SDS PAGE gel separates proteins present in a sample All proteins are covered with negatively charged SDS and migrate according to mass Native PAGE gels run under non-denaturing conditions- SDS and 2-mercaptoethanol are omitted from the gel and sample Proteins separate according to charge, size, shape

  5. IgM serum serum Ig What does a Western blot tell you that a protein gel does not? mAb detects light chain Silver stain Western blot Bromage, E. Comp Biochem Physiol B Biochem Mol Biol. 2006 Jan;143(1):61-9. Epub 2005 Dec 1.

  6. Protein blotting • Two major factors affect the efficiency • The elution from the gel -use the lowest percentage of acrylamide that will allow resolution -high molecular weight proteins blot poorly • Efficiency of binding to the membrane • nitrocellulose (not covalently bound) • Polyvinylidene fluoride (PVDF) • Activated nylon

  7. Transfer of proteins to the membrane

  8. Western blotting-wet transfer apparatus

  9. Western blot-semi-dry transfer of proteins

  10. Detection Primary antibody followed by: Radioactive-labelled 125I staphlococcal protein A or streptococcal protein G Enzyme-linked secondary antibodies -horseradish peroxidase (HRP) -alkaline phosphatase-BCIP/NBT BCIP (5-Bromo-4-Chloro-3'-Indolyphosphate p-Toluidine Salt) and NBT (Nitro-Blue Tetrazolium Chloride). Chemiluminescent detection- HRP catalyzes the oxidation of luminol in hydrogen peroxide. Luminol decays by light emission. AP catalyzes the dephosphyorylation of adamantyl-1-2-dioxetane phosphate, resulting in emission of light.

  11. Can see proteins that are not normally visible

  12. Far western technique Detection of protein-protein interactions using a labelled bait protein

  13. Southwestern blot Figure 7 Distribution of the 52 kDa protein in various mouse tissues as analysed by Southwestern blot analysis Biochemical Journal (1998) 329, 623-629 - www.biochemj.org

More Related