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Preparation of Samples for SDS-PAGE Gel Electrophoresis from Prepared Cell Lysates Priprema uzoraka za SDS-PAGE Gel elektroforeza iz Prepared Stanica Lysates

1. Prepare gels for SDS-PAGE Pripremite gelove za SDS-PAGE
2. Prepare 1X running buffer Pripremite 1X prikazuju tampon
3. Add 50 mL of beta-mercaptoethanol to 950 ml sample buffer (for 1 mL). Dodajte 50 ml of beta-mercaptoethanol do 950 ml sample buffer (1 mL). (only if you have not pre-added beta-mercaptoethanol to sample buffer) (samo ako niste pre-beta-mercaptoethanol dodao da sample 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). Dodaj sample buffer da svaki uzorak (unaprijed odrediti koliko uzorak možete učitati sa dobro - BioRad tankih combs može zadržati o 30uL. Large combs može accomate do 50uL).
5. Vortex samples briefly. Vrtlog uzorci kratko.
6. Poke a hole in cap of each tube (to prevent the tops popping when boiling) Ljenjivac rupa u CAP svaku cijev (da spriječi vrhova iskakanje kada ključanje)
7. Boil in heating block/water bath at (95°C) for 5 minutes (lower temperatures have been shown to prevent non-specific protein aggregation) Kuhati u blok grijanje / na vodenoj kupki (95 ° C) za 5 minuta (niže temperature su pokazala da spriječi non-specifični protein združivanja)

After Boiling Protein Samples - Loading and Running the SDS-PAGE Gel Nakon kipuće protein Uzorci - Loading Trčanje i SDS-PAGE Gel

1. Centrifuge samples for 1 minute at high speed. Centrifuga za uzorke 1 minute i velike brzine.
2. Load sample into each lane. Učitaj uzorak na svaku loptu.
3. Load MW (molecular weight ladder) reference. Učitaj MW (molekularna masa ladder) referenca. (you may want (vi želite svibanj
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) Pokreni gel na 100V (100V kroz slaganje gotovih gel, napon može biti povećana do 200V, kada se izvodi u odvajanje gel s mnogo prikazuju tampon)

Transfer of Protein Samples to Membrane Prijenos proteina na Uzorci Membranski

1. Prepare 1X Transfer buffer Pripremite 1X Transfer tampon
2. Soak and shake gel in Transfer buffer for 15 min Natapanje i stisak ruke gel u tampon-Transfer za 15 min
3. Soak nitrocellulose membrane (or PVDF) and filter paper (extra thick) for several minutes. Natapanje nitrocellulose membrane (ili PVDF) i filtar papir (ekstra guste) za nekoliko minuta.
4. Place sandwich on transfer cell: Mjesto sendvič na transfer stanica:

Extra thick filter paper CLEAR Extra guste filtar papir ČISTO
Membrane Membranski
Gel
Extra thick filter paper BLACK Extra guste filtar papir BLACK

5. Electric transfer: 35V overnite or 90V for 1 hour depending on the size of your protein or your patience! Električni prijenos: 90V ili 35V overnite za 1 sat, ovisno o veličini vaše protein ili strpljenju!

Western Blotting Protocol or Immunoblotting Western bloting Protocol ili Immunoblotting

1. Prepare 1X TBST from stock solutions. Pripremite 1X TBST iz zaliha rješenja.
2. Prepare blocking buffer (3% Bovine Serum Albumin or 5% Blotting-grade milk (Blotto) in TBST). Pripremite se za blokiranje međuspremnik (3% bovini Albumin serum ili 5% upijajući-grade mlijeko (Blotto) u TBST). (you may want to try several different blocking times and conditions). (svibanj želite isprobati nekoliko različitih blokiranje puta i uvjetima).
3. Wash membrane in 1X TBST with shaking for 10 min. Praona membrana u 1X TBST sa potres za 10 min.
4. Block at room temperature for 1 hour with blocking buffer (shaking or circular rotator) Blok na sobnoj temperaturi za 1 sat s blokiranje međuspremnik (potres ili kružne okretaljka)
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). Izleći piliće Vaše membrana (PVDF ili nitrocellulose) sa primary 1 ° Ab antitijela preko noci (za teškim uvjetima, tj. upijajući plastičnost proteina) ili za 1 sat (lako uvjetima kao što je ukupno protein) na 4 ° C (overnite) ili sobnoj temperaturi (1 sat incubation jedina) prekriven saran wrap (blagi potres). You can use plastic tubs from the dollar store for this (use these only for blotting!) or use pipette box bottoms. Možete koristiti plastične kace od dolar spremište za ovu (samo za uporabu tih upijajući!) Ili koristiti pipette okvir dna. The dilution for primary antibody is around 1:1000. The dilution za primarna antitijela je oko 1:1000. See manufacturer's instructions. Pogledajte upute proizvođača.
6. Wash with 1X TBST  3 X 15 min Praona sa 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. Izleći piliće Vaše membrana (PVDF ili nitrocellulose) sa sekundarnog antitijela, 2 ° Ab za 30 - 45 min ili (1 sat - za upijajući teškim uvjetima) na sobnoj temperaturi. The dilution for secondary antibodies is usually 1:10,000 or higher. The dilution za sekundarna protutijela obično 1:10000 ili više. (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. (tj. 1uL srednjih u 10mL od TBST po membrana) Pogledajte upute proizvođača. svibanj Vi želite dodati na vaš antitijelo blokira rješenje za smanjenje ne-posebna obvezujuća pogotovo ako u pozadini visoka je dobio svoj prvi eksperiment.
8. Wash with TBST   4-5 X 10 min. Praona sa TBST 4-5 x 10 min. This is the most important washing step. Ovo je najvažniji korak za pranje.
9. ECL (5min) ECL (5min)
10. Expose to film. Detalji za film. Usually exposure time of western blots is about 10-30 seconds. Obično vremena ekspozicije zapadne blots je oko 10-30 sekundi. Longer (5-10 minutes) for phosphorylation. Više (5-10 minuta) za plastičnost. 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). Ako imate stvarno slab signal, ili vam je potrebno za učitavanje više proteina ili pokušati povećati izloženost vrijeme (do 30 minuta - možete pokušati ostavljajući ga u više kazeta).
11. Keep your membrane! Čuvajte svoje membrana! You can keep your membrane in TBST 1X in the fridge for a while. Možete bi se membrana u TBST 1X u hladnjak na neko vrijeme. 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). Vi svibanj potreba za sljedećih razloga: 1) provjeru učitava (papir recenzentima svibanj pitati za total protein ili učitava standardne kao što su aktinski). Also, you can probe initially for a phospho-protein and then strip and reprobe for total protein. Također, možete istraživati u početku za phospho-protein se svuku, a zatim i reprobe za ukupno protein.

Also see our Western Blotting Membrane Stripping Protocol Također pogledajte naš Western bloting Membranski raskrivanje Protocol