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Samenvatting hoorcollege 1
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Samenvatting hoorcollege 1 - Virusziekten uu
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Virsuziekten samenvatting HC.1Goal: steps you need to take to find out what sec16 does and where
A) Bio-informatics
Bij deze stap krijg je een idee van het moleculaire gewicht en de aanwezigheid van
domeinen. Daarnaast krijg je te zien wat er al bekend is over de functie en lokalisatie (in
verschillende organismen) van het eiwit. Ook krijg je inzicht in welke reagents er
aanwezig zijn (cDNA clone, siRNA antibodies etc.)
NCIB gives information about protein e.g. sequence
ExPASy analysis protein sequence
B) Localisatie/co-localisatie
Antibody
1. cDNA verkrijgen
Buy it
Isolate total RNA make RNA reverse transcribe into cDNA amplify PCR generate
cloning sites
cDNA is DNA dat door reverse-transcriptie uit mRNA is verkregen. cDNA bevat dus geen
introns of signaalsequenties die vaak in een gen te vinden zijn.
2. Eiwit zuiveren en antilichaam verkrijgen
Als je het eiwit wilt gaan zuiveren is het handig als het eiwit een tag heeft zoals GST
of HIS die zich ook in de vector bevind.
Transform bacteria with pGEX-Sec16 (or piece of Sec16) Induce expression with
IPTG Lyse bacteria Pass the extract over a GSH agarose column (GST binds
GSH) Wash extensively Elute the bound protein (GST-Sec16) with GSH
(compete Verify purity by SDS-PAA gelectrophoresis Send your protein for
injection
Soms is het zo dat je het eiwit niet wilt of kan maken. Dan kan je een peptide in het
eiwit selecteren dat geschikt is voor immunisatie (moet hydrofiel zijn). Dit peptide
kan je dan laten maken en vervolgend injecteren bij een muis/konijn/geit.
3. Specificiteit antilichaam bepalen
Check by Western blot (= immuno-blot)
Get a lysate of cells containing your protein
Resolve by SDS-PAGE
Transfer to pvdf membrane
Incubate with your antibody (eg made in rabbit)
Incubate with an anti rabbit antibody coupled to reporter
Detect
Als het goed is krijg je op je western blot één streepje te zien van het juiste
gewicht.
, Hierna moet je nog wel een check doen om er zeker van te zijn dat het bandje je
gewilde eiwit is waar het antilichaam dus aan bindt:
1) Do RNA interference (RNAi) no band visible
When the immune system recognizes a virus by its double stranded RNA, all RNA
molecules with the same sequence are cut into small pieces. Because the RNA is
cut into pieces, protein can no longer be made from it. So if we inject double
stranded RNA with the same sequence as the gene we want to knock down, the
immune system will cut all RNA molecules with that sequence into pieces, even the
ones that are made by the organism itself (see the figure below).
2) Overexpress the protein from plasmid band wordt meer zichtbaar
3) Do antibody incubation on WB in the precence of excess antigen band wordt
minder zichtbaar
4. Eiwit lokaliseren
Nu je weet dat je antilichaam specifiek is, kan je je eiwit gaan localiseren m.b.v.
immunofluorescentie (IF).
fix with paraformalhedyde (or sometimes -20°C methanol migh work) to allow
antibody to go into the cell, you must permeabilize with detergents, such as Triton
X-100, or saponin incubate with the antibody detect with an anti rabbit antibody
coupled to FITC, TR, Alexa, etc
If a pattern is reminiscent of known compartment do co-localisation with a marker of
this compartment using a double labeling strategy with fluorophores of two different
wavelength
Immuno-EM with antibody coupled to gold particles is the way to identify the
compartment on basis of its morphology and to localise the protein of interest at the
same time.
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