Immunoblotting (Western blotting)
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1 Immunoblotting (Western blotting) HIV Transfer to NC membrane membrane gel support buffer Dissociate in SDS Incubate with 1ary antiserum Separate by SDS-PAG Incubate w/ labeled 2nd ab
2 Immunoblotting : overview 1. Preparation of the antigen sample - load sufficient amount of target (immunoprecipitate) - avoid excessive salt and protein load - include positive and negative controls 2. Separation of sample components by electrophoresis - choose the right gel 3. Transfer of the separated polypeptides to a membrane - Nitrocellulose or PVDF - Semi-dry or wet electroblotting 4. Staining the blot for total protein - side strips, prestained standards or biotinylated standards - Ponceau S, India Ink, Coomassie Blue, colloidal gold 5. Antigen detection - block excess binding sites - primary antibody choice - direct vs. indirect methods - background problems (specific vs. nonspecific)
3 SDS-PAG (PolyAcrylamide Gel lectrophoresis)
4 Gel choice
5 lectroblotting systems Tank system transfer membrane support grid support pad filter paper + - electrode Semi-dry system - cathode (black) filter paper gel transfer membrane filter paper + anode (red)
6 Blocking excess protein binding sites
7 Antibody choice for immunoblotting
8 Immunochemical detection systems DIRCT MTHOD INDIRCT MTHOD NC or PVDF blotted antigen molecule labeled primary antibody signal: light or precipitate NC or PVDF blotted antigen molecule l (GH) (cgh) (unlabeled) primary antibody (RacGH) labeled secundary antibody (GaRIg-PO) signal: light or precipitate substrate substrate
9 Immunochemical detection systems DIRCT MTHOD INDIRCT MTHOD NC or PVDF blotted antigen molecule labeled primary antibody signal: light or precipitate NC or PVDF blotted antigen molecule (unlabeled) primary antibody (cgh) (RacGH) labeled ed secundary antibody (GaRIg-PO) signal: light or precipitate substrate substrate A A A A A
10 B nhanced detection systems NC or PVDF blotted antigen molecule (unlabeled) primary antibody biotinlylated secundary antibody (cgh) (RacGH) (biot. GaRIg) labeled (strept)avidin (PO-conjugated (strept)avidin) signal: light or precipitate substrate B B B
11 Labels for use in immunoblotting 125 I - fully quantitative (by gamma counting) - very sensitive - hazardous Horse-radish Peroxidase Alkaline Phosphatase Lanthanide ions (u) - hydrogen peroxide + luminol + film detection - semi-quantitative (by densitometry) - very sensitive - needs darkroom - "blind" development - bromo-chloro-indolyl phosphate + nitroblue tetrazolium (NBT/BCIP) purple precipitate - semi-quantitative (by densitometry) - low background - controlled development - somewhat less sensitive (20x?) - time-resolved fluorescence - quantitative - low background - controlled development - very sensitive - expensive
12 Specificity controls and background control Titrate primary and secondary reagents. Adsorb the primary antibody or even the secondary reagents (e.g. g with an acetone powder of a negative tissue). Compare different blocking solutions. Increase the stringency of the rinses. Negative controls: Omit primary antibody from the detection sequence. This is expecially important when using immunoblotting in combination with immunoprecipitation. Block the primary antibody with the purest antigen available. "Detect" with pre-immune serum from the same animal. "Detect" on a negative control tissue sample. Positive controls: Compare different primary antibodies. Include a positive tissue or a standard of the antigen.
13 Antiserum comparison on the same blot... or reprobe the same blot after "stripping" it.
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