ApplicationNotes | Westernblot:1:250-1:500onmousebrainlysate(unboiledsample)
Immunohistochemistry
Optimalworkingdilutionsmustbedeterminedbytheenduser.
SUGGESTEDWESTERNBLOTPROTOCOL
Solutions
10XTransferbuffer1liter
Tris115.3gm
Glycine24.2gm
[SDS4gm(maybeomitted)]
Fora1Xworkingsolution:1liter
mix700mLwater
200mL
MeOH100mL
10XTransfer
TBSTstock1liter1M
TrispH7.510mL
5MNaCl30mL
10%Tween-205Ml
1.RunSDS-PAGEgelasdesired.
2.SoakPVDFmembrane(ImmobilonPfromMillipore)in100%methanolfor1-2minutestowetthemembrane.
3.Soakmembraneintransferbuffer(seebelow)untilreadytosetupblot(>15min)transfer.
4.Assemblegel-transfersandwichwiththeblottowardthepositiveelectrode.
5.Transferproteinfromgeltomembraneat100voltsfor1-2hoursforsmallmolecularweightproteinsor65mAfor16hours(overnight)forcompletetransferofhighermolecularweightproteins.
6.StainthetransferredbandswithChemiconBLOT-FastStain(CatalogNumber2076).
7.Destainwithdeionizedwater.
8.Blockwith5%non-fatmilk(MarvelorCarnation)inwater,overnightat4°C.Thenon-fatmilkshouldbedissolvedfreshly,centrifuged10,000rpmfor10min,andfilteredthroughglassfilter(GelmanAcrodisc).
9.Incubationwithfirstantibodyovernightwithrockingat4°Cinblockingsolution.Optimalworkingdilutionsandincubationtimewillneedtobedeterminedbytheenduser.
10.Washatleast3x5min.withTBST.Fromthisstage,azideshouldbeomitted.
11.Incubationwiththesecondaryantibody(HRP-conjugatedgoatanti-mouseantibody,forexampleChemiconCatalogNumberAP124P,dilutedappropriately)1hatroomtemperature.
12.Washatleast3x5min.withTBST.
13.PerformECLwithcommercialkit(femtoLUCENT,ChemiconCatalogNumber2078). |