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Western Blotting Protocol Western blotting-protokollen

Western-blot protocol Western-blotting protokol

Preparation of Samples for SDS-PAGE Gel Electrophoresis from Prepared Cell Lysates Forberedelse af prøver til SDS-PAGE gelelektroforese fra Tilberedt Cell Lysates

1. Prepare gels for SDS-PAGE Forbered geler til SDS-PAGE
2. Prepare 1X running buffer Forbered 1X kører buffer
3. Add 50 mL of beta-mercaptoethanol to 950 ml sample buffer (for 1 mL). Tilsæt 50 ml af beta-mercaptoethanol til 950 ml buffer (for 1 ml). (only if you have not pre-added beta-mercaptoethanol to sample buffer) (kun hvis du ikke har pre-tilsat beta-mercaptoethanol at prøve buffer)
4. Add sample buffer to each sample (pre-determine how much sample you can load per well - BioRad thin combs can retain about 30uL. Large combs can accomate up to 50uL). Tilsæt prøve buffer til hver prøve (præ-fastslå, hvor meget prøven kan du indlæse pr godt - BioRad tynde kamme kan bevare ca 30uL. Large kamme kan accomate op til 50uL).
5. Vortex samples briefly. Vortex prøver kort.
6. Poke a hole in cap of each tube (to prevent the tops popping when boiling) Stikke et hul i Cap af hvert rør (for at forhindre toppe popping når kogende)
7. Boil in heating block/water bath at (95°C) for 5 minutes (lower temperatures have been shown to prevent non-specific protein aggregation) Kog til opvarmning blokere / vandbad ved (95 ° C) i 5 minutter (lavere temperaturer har vist sig at forhindre ikke-specifikke protein aggregering)

After Boiling Protein Samples - Loading and Running the SDS-PAGE Gel Efter Boiling Protein prøver - lastning og Running SDS-PAGE gel

1. Centrifuge samples for 1 minute at high speed. Centrifuger prøverne for 1 minut ved høj hastighed.
2. Load sample into each lane. Load prøven i hver vognbane.
3. Load MW (molecular weight ladder) reference. Load MW (molekylevægt stigen) reference. (you may want (du kan evt
4. Run gel at 100V (100V through stacking gel, voltage can be increased up to 200V when running in separating gel with plenty of running buffer) Kør gel på 100V (100V gennem stabling gel, spænding kan øges op til 200V, når du kører i en adskillelse gel med masser af køre buffer)

Transfer of Protein Samples to Membrane Overførsel af Protein prøver til Membrane

1. Prepare 1X Transfer buffer Forbered 1X Transfer buffer
2. Soak and shake gel in Transfer buffer for 15 min Sættetiden og ryste gel i Transfer buffer i 15 min
3. Soak nitrocellulose membrane (or PVDF) and filter paper (extra thick) for several minutes. Sættetiden nitrocellulose membran (eller PVDF) og filtrerpapir (ekstra tyk) i flere minutter.
4. Place sandwich on transfer cell: Sted sandwich forflyttelse celle:

Extra thick filter paper CLEAR Ekstra tyk filtrerpapir CLEAR
Membrane
Gel
Extra thick filter paper BLACK Ekstra tyk filtrerpapir BLACK

5. Electric transfer: 35V overnite or 90V for 1 hour depending on the size of your protein or your patience! Electric overførsel: 35V overnite eller 90V for 1 time afhængigt af størrelsen af din protein eller din tålmodighed!

Western Blotting Protocol or Immunoblotting Western blotting-protokollen eller immunblotting

1. Prepare 1X TBST from stock solutions. Forbered 1X TBST fra stamopløsninger.
2. Prepare blocking buffer (3% Bovine Serum Albumin or 5% Blotting-grade milk (Blotto) in TBST). Forbered blokerende buffer (3% bovint serumalbumin eller 5% blotting-grade mælk (skidefuld) i TBST). (you may want to try several different blocking times and conditions). (du ønsker måske at prøve flere forskellige blokerende tidspunkter og betingelser).
3. Wash membrane in 1X TBST with shaking for 10 min. Vask membranen i 1X TBST med rystes i 10 min.
4. Block at room temperature for 1 hour with blocking buffer (shaking or circular rotator) Block ved stuetemperatur i 1 time med blokerende buffer (ryster eller cirkulær rotator)
5. Incubate your membrane (PVDF or nitrocellulose) with primary 1° Ab antibody overnight (for difficult blotting conditions ie phosphorylation of proteins) or 1 hour for ( easy conditions such as total protein) at 4°C (overnite) or room temperature (1 hour incubation only) covered with saran wrap (gentle shaking). Inkubér din membran (PVDF eller cellulosenitrat) med primær 1 ° Ab antistof natten over (for vanskeligt blotting betingelser dvs fosforylering af proteiner) eller 1 time for (let betingelser såsom total protein) ved 4 ° C (overnite) eller stuetemperatur (1 time inkubation kun) er dækket med saran wrap (svag rysten). You can use plastic tubs from the dollar store for this (use these only for blotting!) or use pipette box bottoms. Du kan bruge plastic baljer fra dollar-butik for denne (brug disse kun for trækpapir!) Eller bruge pipette boksen bottoms. The dilution for primary antibody is around 1:1000. Fortyndingsfaktoren for primære antistof er omkring 1:1000. See manufacturer's instructions. Se fabrikantens anvisninger.
6. Wash with 1X TBST  3 X 15 min Vask med 1X TBST 3 X 15 min
7. Incubate your membrane (PVDF or nitrocellulose) with secondary antibody, 2° Ab for 30 – 45 min or (1 hour - for difficult blotting conditions) at room temperature. Inkubér din membran (PVDF eller nitrocellulose) med sekundære antistof, 2 ° Ab i 30 - 45 min eller (1 time - for vanskeligt blotting betingelser) ved stuetemperatur. The dilution for secondary antibodies is usually 1:10,000 or higher. Fortyndingsfaktoren for sekundære antistoffer er sædvanligvis 1:10000 eller højere. (ie 1uL of secondary in 10mL of TBST per membrane) See manufacturer's instructions.  You may want to add your antibody to blocking solution to decrease non-specific binding especially if you got high background in your first experiment. (dvs. 1uL af sekundær i 10 ml af TBST pr membran) Se fabrikantens anvisninger. Du kan tilføje dine antistof mod blokering løsning at falde uspecifik binding især hvis du fik høj baggrund i dit første eksperiment.
8. Wash with TBST   4-5 X 10 min. Vask med TBST 4-5 X 10 min. This is the most important washing step. Dette er den vigtigste vask skridt.
9. ECL (5min) ECL (5min)
10. Expose to film. Expose til film. Usually exposure time of western blots is about 10-30 seconds. Normalt udsat tidspunktet for western blotting er omkring 10-30 sekunder. Longer (5-10 minutes) for phosphorylation. Længere (5-10 minutter) for fosforylering. If you have a really weak signal, either you need to load more protein or try to increase exposure time (up to 30 minutes - you can try leaving it in a casette longer). Hvis du har en virkelig svagt signal, enten du har brug for at indlæse mere protein eller forsøge at øge eksponeringen tid (op til 30 minutter - du kan prøve at forlade det i en kassette længere).
11. Keep your membrane! Hold dine membran! You can keep your membrane in TBST 1X in the fridge for a while. Du kan holde din membranen i TBST 1X i køleskabet i et stykke tid. You may need it for the following reasons: 1) checking loading (paper reviewers may ask for total protein or a loading standard such as actin). Du kan have brug for det af følgende årsager: 1) kontrol lastning (papir kontrollanterne kan anmode om total protein eller en lastning standard såsom actin). Also, you can probe initially for a phospho-protein and then strip and reprobe for total protein. Du kan også sonden i første omgang for en phospho-protein og derefter strimler og reprobe for total protein.

Also see our Western Blotting Membrane Stripping Protocol Se også vores Western blotting Membrane Stripning protokol