Molecular Techniques and Methods

Western Detection of Protein

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

  • This procedure is adjusted for Immobilon-P (PVDF membrane, Millopore).



    MATERIALS AND SOLUTIONS

    Transfer Buffer (1 L)
     25 mM Tris-HClpH 8.3 --------------------------------- 25 ml of 1M Tris-HCl
    192 mM Glycine -------------------------------------- 14.4 g
    20 % Methanol --------------------------------------- 200 ml
    DW --------------------------------------------------- 760 ml
  • Keep at 4oC.
  • Do not reuse Transfer Buffer.
  • SDS eluted from SDS-PAGE coats the protein, resulting linearized-protein passing through the pore.

    Ponceau-S red Solution (50 ml)
     0.5% Ponceau-S red --------------------------------- 0.25 g
    1 % Acetic acid --------------------------------------- 500 ul
    DW --------------------------------------------------- 49.25 ml

    1% BSA-Blocking Buffer (200 ml)
     1% BSA -------------------------------------------- 2 g BSA
    1x TBS (or 1x )PBS -------------------------------- 20 ml of 10x TBS
    0.05 % Tween-20 ----------------------------------- 200 ul of 50% Tween-20
    DW ------------------------------------------------ 180 ml
  • For detection of phosphorylated protein, use TBS. Phosphate in PBS may interfere antibody binding.

    3% BSA-Blocking Buffer (200 ml)
     3% BSA -------------------------------------------- 6 g BSA
    1x TBS (or, 1x PBS) ------------------------------- 20 ml of 10x TBS
    0.05 % Tween-20 ----------------------------------- 200 ul of 50% Tween-20
    DW ------------------------------------------------ 180 ml
  • For detection of phosphorylated protein, use TBS. Phosphate in PBS may interfere antibody binding.


    1% NFM-Blocking Buffer (200 ml)
     1% NFM (non fat milk) ----------------------------- 2 g
    1x TBS (or, 1x PBS) ------------------------------- 20 ml of 10x TBS
    0.05 % Tween-20 ----------------------------------- 200 ul of 50% Tween-20
    DW ------------------------------------------------ 180 ml

    5% NFM-Blocking Buffer (200 ml)
     5% NFM (non fat milk) ----------------------------- 10 g
    1x TBS (or, 1x PBS) ------------------------------- 20 ml of 10x TBS
    0.05 % Tween-20 ----------------------------------- 200 ul of 50% Tween-20
    DW ------------------------------------------------ 170 ml

    1x Wash Solution (1 L)
     1x TBS -------------------------------------------- 100 ml of 10x TBS
    0.05 % Tween-20 ----------------------------------- 1 ml of 50% Tween-20
    DW ------------------------------------------------ 900 ml

    Mild Stripping Buffer (100 ml)
     Glycine --------------------------------------------- 1.5 g
    0.1% SDS -------------------------------------------- 1 ml of 10% SDS
    Tween-20 -------------------------------------------- 1 ml
    DW -------------------------------------------------- 96.5 ml

    Harsh Stripping Buffer (100 ml)
     62.5 mM Tris-HClpH6.8 ----------------------------- 6.4 ml of 1 M Tris-HClpH6.8
    2% SDS -------------------------------------------- 20 ml of 10% SDS
    0.1M 2-ME ----------------------------------------- 0.8 ml of 2-ME
    DW ------------------------------------------------- 72.8 ml

    10x TBST (200 ml)
     200 mM Tris-HClpH7.4 ------------------------------- 40 ml of 1 M Tris-HClpH6.8
    1.5 M NaCl ------------------------------------------ 60 ml of 5 M NaCl
    27 mM KCl ------------------------------------------ 5.4 ml of 1 M KCl
    DW -------------------------------------------------- 96.4 ml

    ECL Detection Reagents
     a) Luminol stock: Luminol (Sigma) 4mg/ml in DMSO
    b) Enhancer stock: p-iodophenol (Aldrich) 1 mg/ml DMSO
    c) Tris Buffer: 100mM Tris.HCl pH7.5
    d) 3% H2O2

  • Stock solns a) and b) should be kept in -20oC for <6 months.
  • Working soln. (Prepare just before use)
    a) 0.5ml, b) 0.5ml, c) 2.5ml, d) 25ul, ddw 1.5ml (final 5ml)



    PROCEDURES

    Transfer
    1. Soak the SDS-PAGE gel in Transfer Buffer to remove SDS.
  • 15 min for 0.75 mm thick gel.
  • 20 min for 1 mm thick gel.
  • 30 min for 1.5 mm thick gel.
  • SDS-coated linearized protein can pass through the pore in the membrane.

    2. For each gel, cut the blotter paper and membrane to fit the transfer cassette.
  • Wet a PVDF in 100% methanol.
  • Wet a nitrocellulose membrane with distilled water.

    3. Soak all membrane types in DW for 2 minutes.

    4. Soak all membrane types in Transfer Buffer for 5 minutes.

    5. Assemble the transfer sandwich under buffer to minimize trapping air bubbles.
  • Wet each component of the sandwich cassette in the transfer buffer before assembly.

    6. Assemble the sandwich components in the following order:
  • Clear side of the cassette (facing (+) electrode)/ sponge/ 1 piece of blotter paper/ membrane/ gel/ 1 piece of blotter paper/ sponge/ black side of the cassette (facing (-) electrode).
  • As each layer is added, take great care to remove all air pockets by rolling a clean pipet or test tube over the layer.

    7. Fill tank with ice-cold Transfer Buffer to a level that covers the electrode panels and put stir bar in bottom.
  • Insert the cassette in the proper orientation (gray side facing the positive electrode).
  • The buffer should cover the electrode panels by at least 2 cm once the transfer cassettes are in place, but should not touch the base of the banana plug.

    8. Place tank on a magnetic stirrer.

    9. Connect the power supply to the transfer tank and transfer the proteins to the membrane under the conditions as follow.
  • For 1 hour transfer: electrical conditions - 100 V, 300 mAmp (at the beginning).
  • Do not do O/N transfer at low volt. Gel sticks to the membrane.

    10. Throughout the transfer, stir the buffer with a magnetic stirrer.
  • The cooling system set to maintain 15 to 20oC must be used in transfer procedures longer than 30 minutes to prevent overheating.

    11. Turn off, and disconnect from the transfer unit.

    12. Remove transfer cassette and disassemble transfer stack.
  • Place the gel in Coomassie blue stain to verify transfer.
  • Stain the membrane with Ponceau S (below) to determine position of protein markers.
  • Any stained protein remaining in the gel indicates incomplete transfer.

    13.Do not dry membrane.
  • Optional, if needed, dry membrane as follow;
  • Air dry the membrane for 5-10 min.
  • Soak the membrane in 100% methanol for 10 seconds.
  • Air dry the membrane on filter paper for 15 min.

    14. For dried PVDF membrane, place in 100% methanol, then in distilled water to wet.

    15. Optional, Confirm that the protein is efficiently binding to the membrane by staining with Ponceau S or lndia ink.
  • Stain the blot in a solution of 0.5% Ponceau-S red Solution for 1 min.
  • Destain the blot in DW to desired contrast.
  • 0.1 N NaOH removes Ponceau-S stain completely.


    Hybridization
  • Continue hybridization with proper antibody.

    16. Block the membrane in 3% BSA-Blocking Buffer for 30 min-1 hr at RT on rocker.
  • Do not block >1 hr. Blocking also blocks the target protein.
  • If non-specific background observed, block in 5% NFM-Blocking Buffer for 20 min.
  • Usually, 3% BSA is enough for blocking.

    17. Incubate the blot with primary antibody in 1% BSA-Blocking Buffer for 1 hr - 2 hr at RT, or O/N at 4oC on rocker.
  • Multiple antibodies can be hybridized at the same time.

    18. Wash the blot in 1x PBS for 3-5 min.

    19. Incubate blot with secondary antibody in 1% NFM-Blocking Buffer for 30 min - 1 hr at RT on rocker.
  • Secondary antibody = 1:10,000-100,000 dilution
  • DO not incubate with 2nd Ab for >1 hr. Enzyme activity goes down as time pass.

    20. Rinse the blot in 1x Wash Solution for 5 min.
  • If signal is weak or not detected, wash membrane with PBS without Tween-20.

    21. For secondary antobody-peroxidase, add Pierce SuperSignal (two solutions, mix 3 ml of each per blot).
  • SuperSignal West Pico Stable Peroxide Solution (Pierce)
  • SuperSignal West Pico Luminol Enhancer Solution (Pierce)

    22. Incubate RT for 3 min.

    23. Pick up the blot and remove excess solution.

    24. Autoradiograph for 10 seconds to 60 min.


    Primary
    antibody
    Source
    Conc.
    Incubation
    Secondary antibody
    Conc.
    Exposure
    Size
    (kDa)
    Actin
    Lab Vision
    1:2000
    O/N, 4oC
    Mouse
    1:10,000
    1 sec
    42
    Tubulin
    Santa Cruz
    1:500
    O/N, 4oC
    Mouse
    1:10,000
    1-3 min
    55
    c-fms
    Santa Cruz
    (sc-692)
    1:100
    O/N, 4oC
    Rabbit
    1:10,000
    1 hr
    135 and 165
    CSF-1
    Abcam (ab66236)
    1:100
    O/N, 4oC
    Mouse
    1:10,000
    1 hr
    45, 55, 65, and 200
    HuR
    Santa Cruz
    1:500
    O/N, 4oC
    Mouse
    1:10,000
    5 sec
    34
    Vigilin
    Santa Cruz
    1:250
    O/N, 4oC
    Mouse
    1:10,000
    1 hr
    155
    Nucleolin (C23)
    Santa Cruz
    1:500
    O/N, 4oC
    Mouse
    1:10,000
    1-5 min
    75 and 110
    Nucleophosmin (B23)
    Santa Cruz
    1:500
    O/N, 4oC
    Mouse
    1:10,000
    1-5 min
    33
    GRP75
    Santa Cruz
    1:1000
    O/N, 4oC
    Rabbit
    1:10,000
    30 min
    75
    HDAC1
    Santa Cruz
    1:500
    O/N, 4oC
    Mouse
    1:10,000
    1 min
    50
    TTP
    Santa Cruz
    1:100
    O/N, 4oC
    Goat
    1:10,000
    30 min
    44
    FBP2
    Santa Cruz
    1:100
    O/N, 4oC
    Goat
    1:10,000
    30 min
    74
    hnRNP a/b/c/d
    Santa Cruz
    1:500
    O/N, 4oC
    Rabbit
    1:10,000
    1 min
    37-50
    BRF1
    Santa Cruz
    1:100
    O/N, 4oC
    Goat
    1:10,000
    30 min
    100



    Stripping and Reprobing
    While stripping , sample proteins will be lost.

    Mild stripping condition
    25. Put the membrane in a seal-a-meal bag with 10-15 ml Mild Stripping buffer.

  • Incubate at RT for 5-10 min. 2x.
  • Wash in PBS for 10 min. 2x
  • Wash in TBST for 5 min. 2x.
  • Ready for blocking.

    Harsh stripping condition
    26. Put the membrane in a seal-a-meal bag with 10-15 ml Harsh Stripping buffer.

    27. Incubate the membrane in the 50ºC water bath rocker for 20-40 minutes.

    28. Rinse the membrane under running water for 1-2 hrs.
  • Traces of 2-mercaptoethanol will damage the antibodies.

    29. Wash for 5-10 min in TBST.

    30. Ready for blocking.




    NOTES

  • No signal problem - Most often over-blocking is the cause. Use less NFM. Remember blocking also blocks target protein.
  • For detection of low abundant protein, BSA is better than NFM.

  • PVDF (Polyvinylidene Difluoride) membranes
    PVDF membranes are hydrophobic with a pore size of 0.2 or 0.45 um. Proprietary alterations in the Pierce PVDF membrane make it more resistant to discoloration which often occurs with other commercially available PVDF membranes. These membranes have high binding affinity for proteins and nucleic acids and may be used for applications such as Western, Southern, Northern and dot blots. The PVDF membranes offer a better retention of adsorbed proteins than other supports including nitrocellulose.

  • Nitrocellulose Membranes
    Nitrocellulose membranes are the most popular matrix used in protein blotting. All of the following are suitable for Western blotting. Most proteins can be successfully blotted using a 0.45 um pore size membrane. For proteins of low molecular weight or peptides, a 0.2 um pore size membrane is recommended.
    Nitrocelluloses membranes are the most popular membranes for Western, Southern and Northern Blotting. The membranes bind both proteins and nucleic acids. Nitrocellulose exhibits high binding capacity and has low background. See also: Electroblotting Nitrocellulose (Cellulose nitrate-) Membrane, pore size 0.45 mm, plain, white.
  • Nitrocellulose-Membrane, pore size 0.2 mm, white
    Especially for use with proteins of low molecular weight (< 20 000 Dalton): Burnette, N. (1981) Anal. Biochem.112, 195-203; Tsang, V.C.W. et al. (1983) Methods Enzymol. 92, 377

  • Nylon-Bind and Fluorobind-Membranes
    Nylon-Bind Membranes feature low background, high sensitivity and high binding capacities for blotting of proteins andnucleic acids. The high inner surface of Nylon-Bind membranes is based on the unique microporous structure of the nylon material.
    Fluorobind Membranes are based on PVDF-type chemistryand are suited especially for protein blotting and protein sequencing.

  • The gel will shrink in Transfer Buffer and, if this occurs during transfer, smeared patterns will result.
    Equilibration preshrinks the gel to prevent this.
    If the gel was run in a buffer system that is very different from the Transfer Buffer it is important to equilibrate it longer (IS minutes).
    But if a low acrylamide gel or low molecular weightproteins (< 40 kD) are used, they may diffuse out of the gel if it is equilibrated too long.

  • In general, the proteins are negatively charged due to the bound SDS and will transfer from the cathode (-, black) to the anode (+, red).
    Some proteins may be positively charged at the transfer pH and will move in the other direction toward the negative electrode.
    Placing an extra sheet of membrane on the other side (-) of the gel will bind the positively charged proteins.

  • Transrer efficiency depends on transfer time, applied voltage, protein size, the percent acrylamide, and the thickness of the gel.
    If transfer is incomplete, decrease the percentage acrylamide so that the protein moves about halfway down the gel.
    This ensures a complete transfer. Buffer composition is also an important factor.
    Typical Transfer Buffer contains 20% methanol, which improves protein binding to nitrocellulose but can inhibit transfer.
    SDS is frequently added to improve transfer, particularly of larger proteins.
    Unfortunately, SDS inhibits protein binding to the membrane. Optimization of the SDS and methanol concentrations for difficult-to-transfer proteins is largely empirical.

  • In general, thin (0.75 mm), low % acrylamide gels transfer most efficiently.
    If transfer is not complete, try a lower %T and/or a thinner gel.
    Longer transfer times (e.g., double) may also help.
    For particularly difficult-to-transfer proteins (eg., larger than 80 kD), add SDS, or leave out or reduce the amount of methanol.
    Staining the gel after transfer will give an indication of transfer efficiency.

  • Any protein passing through the membrane is visible as staining on the back of the membrane.
    SDS in the transfer buffer or old nitrocellulose can cause poor protein binding.




    KIT INFORMATION




    REFERENCES

  • ECL reference - Schneppenheim et al (1991) Electrophoresis 12:367-372

  • Blotting tips


  • Please send your comment on this protocol to "editor@MolecularInfo.com".
    		•
    Home
    MT&M
    Online Journal
    Hot Articles
    Order Products
    Classified