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General Western Blotting protocoldoc

By Eric Olson,2014-06-06 10:26
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General Western Blotting protocoldoc

    General protocol for Western Blotting (AP and HRP conjugates)

     (last updated 3-23-07)

    By : Fang Yi

BUFFERs and Solutions:

    Transfer buffer (1.5L, always prepare fresh before transfer):

    ; 50 mM Tris, 39 mM Glycine, 20% methanol (v/v), pH 8.0

    ; Methanol slows down the transfer, add SDS to larger protein to speed up the

    transfer

20X TBS stock (no Tween 20) (200 ml)

    ; 200 mM Tris, 3M Nacl, pH 8.0

1X TBST (prepare right before the analysis) (1L)

    ; 10 mM Tris, 150 mM NaCl, pH 8.0, 0.1% Tween

Blocking buffer:

    ; 1X TBST with 5% non-fat milk

For AP conjugates:

    TSM

    ; 100 mM Tris, 100 mM NaCl, 50 mM MgCl2, pH 9.5

    For development:

    ; BciP, NTB, TSM

For HRP conjugate (colorimetric only):

    10X Tris-saline:

    ; 9% (w/v) NaCl in 1 M Tris-HCl, pH 8.0

    Staining solution for HRP:

    ; 18 mg 4-chloro-1-naphthol (sigma cat#: C8890) in 6 ml methanol. Add 24 ml

    1X Tris-saline followed by 600 ul 3% hydrogen peroxide (final 0.2% H2O2)

Procedures:

    1. SDS-page Gel:

    ; Run a protein gel to separate the proteins, runs it until the bromophenol blue

    front reaches the end of gel.

    ; also load a protein marker to use as a control for protein transfer.

    2. During gel running, prepare:

    ; PVDF membrane:

    Cut appropriate size membrane, and activate it by:

    ; Soak in 100% methanol for 10 sec. (PVDF is hydrophobic)

    ; Soak in ddH2O for 30 sec

; Soak in transfer buffer for >10 minutes

    Whatman paper filter & sponges

    ; Soak cut filters (whatman paper) and sponges in transfer buffer for 10 minutes

    (push bubbles out)

    ; (don’t soak the gel in transfer buffer if you are transferring small proteins); but

    soaking in transfer buffer for short time (eg. 3 minutes) can wash salt off gel to

    reduce current during transfer.

3. Assemble the transfer apparatus in the order of

    Black(negative) Sponge---Filter---Gel---PVDF memberanefilter---spongeRED

    end

    4. Ensure the packing is tight and the chamber does not leak. ( if you press the sponges

    to the inner side, you should see the solution levels)

    5. Transfer at RT (ice in apparatus) for 120 minutes at constant current (350 mA)

    ; This setting may need to be optimized for diff. size of proteins. Small

    proteins (<20 kD) tends to transfer faster, larger protein might need

    longer transfer time

    ; The setting that has worked for me (working with proteins with diff. sizes

    from 37kD to 180kD) is: total work ( W) = I*V*time ~ 15 . For example,

    if using a constant voltage of 40 volts, current is 0.2 A, then a time setting

    of 2 hrs (40*0.2*2=16) usually is enough for complete transfer as judged

    by the marker.

    6. Disassemble the chamber. To ensure your protein is transferred before all the

    following steps, commassie blue stain the SDS-gel to estimate the transfer efficiency 7. Block the membrane for 1 hr in blocking buffer: 5% (w/v) non-fat milk in TBST. 8. Wash membrane for 3* 5 min w/ TBST;

    9. Incubate membrane in Primary antibodies diluted in blocking buffer (TBST) at 4

    deg overnight and 1 hr at room temperature.

    10. Wash for 3*5 min w/ TBST; nd11. Incubate with 2 Ab for 1 hr at RT in TBST

    12. wash for 2*5 min w/ TBST

    13. wash 1*5 min w/ TBS nd14. wash 2*10 min/TSM (if using AP conjugated 2 Antibody)

    15. Development:

    a) Colorimetric: Stain with AP or HRP staining solution until the signal is clearly

    visible nd; For AP-conjugated 2 Ab, add 33 ul BciP , 42 ul NBT in 10 ml TSM, pH 9.5

    (5-15 minutes)

    ; For HRP: Staining solution for HRP: 18 mg 4-chloro-1-naphthol (sigma cat#:

    C8890) in 6 ml methanol. Add 24 ml 1X Tris-saline followed by 600 ul 3%

    hydrogen peroxide (final 0.2% H2O2) (1-5 minutes)

    ; at RT for 5~15 minutes until the band shows up; (Don’t shake blots during

    color development)

    b) ECL solution followed by Film Exposure:

    1. Prepare ECL reagents: mix the two reagents 1:1 (~1ml per blot) and pipette

    enough vol on a piece of Saran wrap taped to the bench.

    2. Drag the blot along the edge of the tray, drain excess TBST, then place the blot

    with protein side down on the ECL solution.

    3. Incubate for 1 min then drain excess reagent and transfer the blot to a plastic

    report cover.

    4. Expose immediately (few seconds 1 hr).

    16. Stop solution: 20 mM Tris, pH 7.4, 5 mM EDTA OR rinse twice in water.

    17. Dry the membrane and expose ASAP.

     thTIPs from the Millipore Protein Blotting Handbook (5 version):

To reduce persistent background: nd; Use High salt wash after the 2 Ab incubation (PBS or TBS with 0.5 M NaCl and

    0.2% SDS)

    ; Incubate the membrane in high salt buffer for 30 minutes w/ gentle shaking

    ; Rinse blot with Milli-Q water and proceed as usual

To reduce overall high background: nd Use higher dilution of the 2 Ab

To reduce high non-specific signal: st Use higher dilution of the 1 Ab and lower protein yield.

To view the protein on the membrane:

    Soak in 20% methanol, watch on the light box

Role of Tween:

    0.05% -0.1% Tween can help renature the antigen, thus increasing improved recognition of specific antibodies; but too high % of Tween can wash away blotted proteins

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