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        Cell Lysis/Western/IP

        互联网

        3306

        Cell Lysis/Western/IP

        Steven Finkbeiner,Departments of Neurology and Physiology,UCSF

        compiled by 6/95 by AJS from AB)Fresh 1X Lysis

        Fresh 1X LysisFinal5ml10ml
        2X Lysis 2.5 ml5ml
        NaCl0.25M0.25ml of 5M0.5 ml of 5M
        Na3VO40.1mM25l (μl) of 200 mM5l (μl) of 200mM
        NaF50mM0.5 ml of 0.5M1ml of 0.5M
        DTT1mM5l (μl) of 1mM10l (μl) of 1mM
        Aprotinin3ug/ml15l (μl) of 1 ug/μl3l (μl) of 10 ug/μl
        Pepstatin2 ug/ml10l (μl) of 1 ug/μl20l (μl) of 1 ug/μl
        Leupeptin1 ug/ml5l (μl) of 1 ug/μl1l (μl) of 10 ug/μl
        Okadaic 1:500
        PMSF 35l70l
        H2O 1.75 ml3.5ml

        1X Lysis Buffer -- 4℃

        Cool eppys -- 4℃ put 4x # of tubes needed

        PBS -- 4℃

        Stimμlation -- 37℃ 10' NGF=100ng/ml à first aspirate 5 ml!

        KCl=5 ml to 10 ml

        Lyse -- on ICE TRAY Stop stimμlation

        asp.Media/wash 2XPBS/ +500l lysis buffer

        scrape into eppy

        incubate 45' 4℃ à Vortex several times during

        spin 10 secs 4℃

        divide sample: ~400 l for IP/ ~ 100 l for Western

        IP -- 1° Ab => 4℃ 1 hour rocking

        if 1° is not rab.à 2° Ab => 4℃ 1 hour rocking (RaM 2l)

        -- PrA/Seph 4℃ 1 hour rocking 40l

        -- Spin down 4℃ 1'

        -- Wash 3X: 2X Lysis Buffer (~500 l)/ 1X PBS

        Sample Prep -- (a)IP: add (40 l)2X Sample Buffer (1X V PrA/S)

        -- (b)W: add (50 l)3X Sample Buffer

        Boil 5'

        Run Gel -- 100 V through Stacking

        100 00 V after (in Separating)

        Prepare Transfer -- make fresh TB: 1L= 200ml 5X TBb

        200 ml Methanol

        600 ml H20

        -- cut fresh nitrocellμlose (11 X 17 cm)& Whatman soak in TB

        -- 2 grids outside plate on Yellow Tape side

        1 grid in middle SMOOTH SIDE TO GEL!

        Cushions

        pour in TB until just wet

        put 1 Whatman over Gel à smooth out

        place onto cushion

        Nitrocellμlose over gel

        2nd Whatman over Nitrocellμlose à smooth out bubbles

        4 cushions on top

        2nd Grid SMOOTH SIDE TO GEL!

        Add more TB

        2nd metal plate

        place in tank: liquid shoμld cover gel area Transfer use black battery charger: 24V 45'

        After Transfer remove blot and place in container face up quick rinse TBST

        Block 3% BSA/TBST or 5% Milk/TBST 1 hour RT shaking

        1 °Ab dilute in 20ml: 3% BSA/0.05% NaN3/TBST

        Wash pour 1 ° Ab back (RE-USE!)quick rinse,then 3X5' rinse TBST ° Ab HRPaM or HRPaR (depending on 1° Ab)dilute 1:20000 (1.25 l in 25 ml): 3% BSA/TBST 1 hour RT shaking

        Wash quick rinse,then 3X5' rinse TBST

        ECL 5 ml DuPont NEN White + 5 ml Dark bottle place blot into solutin for 1' + agitation put onto Saran Wrap and smooth out bubbles put fluorescent dot markers on for orienta tion

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