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Western Blotting/Immunoblotting (WB/IB) Protocol

Protocols & Procedures

Tips and Tricks

Reconstitution & Storage Instructions  
Western Blotting/Immunoblotting (WB/IB) Protocol  WB/IB Tips & Tricks
Immunohistochemistry (IHC) Protocol  IHC Tips & Tricks
Immunocytochemistry (ICC) Protocol  ICC Tips & Tricks
Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol  ELISA Tips & Tricks
Blocking Peptide Competition Protocol (BPCP)  
Immunoprecipitation (IP) Protocol  IP Tips & Tricks
Antibody Array (AA) Protocol 

 

Western Blotting / Immunoblotting (WB / IB)

Description: 

A western blot is a technique used to identify the presence of an antigen in a particular tissue homogenate or protein extract.  Typically, protein samples are resolved by their size by gel electrophoresis and transferred onto a membrane.  The membrane is then probed with an antigen specific antibody which is then itself detected using an enzyme conjugated antibody.  Enzyme substrate is then applied and the membrane is visualized for the presence of signal.  see example western blots from our validation program!

Procedure:

Please consult the product data sheet for the appropriate concentration of primary antibody and any other special conditions.


Cell Lysate Preparation:

A. Reagents and Materials

  • Phosphate Buffered Saline; 1X PBS, pH 7.4
  • SDS-Loading Buffer: 0.05M Tris-HCl, pH6.8, 0.1M DTT, 2%SDS, 10% glycerol, 0.1% bromophenol blue
  • Cell Detaching Trypsin Buffer: 0.25% trypsin in 0.53M EDTA solution
  • Cell Lysis Buffer (1% NP40, 0.5% DOC, 1mM EDTA , 65mM Tris-HCl pH=6.4, 1mM PMSF, 1ug/ml Apoprotin, 1ug/ml Leupeptin, 1ug/ml Pepstatin)
  • BCA Protein Assay Kit

B. Equipment

  • Ultrasonicator

C. Protocol

    • Cell culture and harvest
      a) Cell subculture
          i) Suspension cells: spin down cells and re-suspend pellet in RPMI 1640 with 10% FBS to make the final concentration around 105 cells/ml, and incubate at 37C, 5%CO2.

          ii) Adherent cells: digest adherent cells in Cell Detaching Trypsin Buffer. Then spin down cells and re-suspend the pellet in DMEM with 10% FBS to make the final concentration around 105 cells/ml, and incubate at 37C, 5%CO2 .

      b) Incubate cells for about three days and count the cells. When the concentration reach 106 cells/ml, detach cells, spin down and wash twice with 1XPBS .
      c) Add cell lysis buffer to cell extracts (1ml lysis buffer /5×107 cells). Vortex the mixture for 5min and store at -20C.
    • Preparation of tissue extracts
    • a) Thaw the frozen, raw tissue sample by vortexing and repeat the freeze-thaw cycle twice.
        b) Disrupt tissue with ultrasonicator.  Keep the entire operation on ice.
    • Measure concentration of cell samples
    • a) Transfer contents to a microcentrifuge tube. Centrifuge at 12,000 rpm for 15 minutes. Then collect supernatant into an appropriately labeled tube (for the detection of membrane bound proteins, use the insoluble, cell pellet). Dilute the supernatant lysate and the whole lysate without centrifugation at 1:4, 1:8 and 1:16 with 1XPBS. Measure protein concentration using cell lysis-compatible protein assay (BCA protein assay).
      b) Mix the cell lysate with SDS loading buffer to make the desired final concentration. Incubate the lysates at 100°C for 10min.
      • Quality control
      • a) Test cell lysate by SDS-PAGE. The cell lysate is evaluated as qualified, if the bands are clear and have no obvious smear.
      b) Test cell lysate by Western Blot. The primary antibody we used is the antibody against the marker proteins. The cell lysate is evaluated as qualified, if the WB image shows five bands.

SDS-PAGE

A. Reagents and Materials

  • Stacking gel buffer: 0.5M Tris-HCl, pH6.8, 0.4% SDS
  • Separating gel buffer: 1.5M Tris-HCl, pH8.8, 0.4% SDS
  • Acrylamide stock solution: 29% acrylamide plus 1.0% bis-acrylamide
  • 10% ammonium persulfate
  • TEMED(N,N,N’,N’-tetramethylene-ethylenediamine)
  • Electrophoresis buffer: 0.25M Tris Base, 2M Glycine, 1% SDS
  • Coomassie gel stain solution: 0.1% Coomassie blue R-250, 30% ethanol, 10% acetic acid.
  • Coomassie gel destain solution: 30% ethanol, 10% acetic acid.

B. Equiment

  • Minigel apparatus: Bio-Rad Mini-Protean 3 Dodeca Cell
  • Power supply: Biio-Rad PowerPac HC
  • Gradient gel former: Bio-Rad Model 485 Gradient Former

C. Protocol

  • Assemble mutil-casting chamber according to the manufacturer’s instructions. All following operations related with apparatus should follow manufacture’s instructions.
  • Make separating gel as following formula in a suitable beaker. Ammonium persulfate and TEMED should be added before pouring gel.
  • a) For making 12 linear slab gels (ml)
Separating concentration8%10%12%15%
Separating gel buffer 16 16 16 16
Acrylamide stock solution 16 20 24 30
H2O 27.4 23.4 19.4 13.4
10% ammonium persulfate 0.6 0.6 0.6 0.6
TEMED 0.05 0.05 0.05 0.05

b) For making 12 gradient gels (ml):

Separating concentrationLowHigh
6% 10% 18% 20%
Separating gel buffer 8 8 8 8
Acrylamide stock solution 6 10 18 20
H2O 15.5 11.7 3.7 1.6
10% ammonium persulfate 0.3 0.3 0.3 0.3
TEMED 0.025 0.025 0.025 0.025

Use gradient gel former to make gradient gel. Use 10-20% gel for low molecular weight (MW< 20kD) protein identification and use 6-18% gel for high molecular weight (MW>100kD) protein identification.

  • Pour the separating gel
  • a) After adding ammonium persulfate and TEMED immediately mix the gel solution gently and carefully introduce solution into gel casting chamber. Stop the pouring when gel solution reach to about 6 cm height and layer about 0.5 ml isobutanol on top of the separating gel solution to keep gel surface flat. Allow gel to polymerize within 10-30 minutes.
  • Pour the stacking gel
  • a) When the gel has polymerized a distinct interface will appear between the separting gel and the isobutanol. Drain isobutanol and wash the surface with distill water. After adding ammonium persulfate and TEMED immediately mix the gel solution gently and carefully introduce solution onto separating ge until solution reach top of front plate. Carefully insert comb into gel until bottom of teeth reach top of front plate. Allow gel to polymerize within 10-30 minutes.
  • Loading samples
  • a) After stacking gel has polymerized, remove comb carefully and place the gel into electrophoresis chamber. Add electrophoresis buffer to inner and outer reservoir. Introduce sample solution, including molecular weight standard, into well (20-25ug total cell lysate/homogenate, unless otherwise specified).
  • Running gel
  • a) Cover the lid and attach electrode plugs to proper electrode. Turn on power supply to 80V and run about 30 minutes. Change voltage to 160V when samples enter the stacking gel and form single narrow line. Stop electrophoresis when dye front migrate to the bottom of the gel in about 60 minutes. Remove electrode plugs from electrodes and gel plates from assembly.
  • Processing Gel for different uses
    a) Staining gel with Coomassie Blue.  If separated proteins are needed to be seen in the gel directly, stain gel with Coomassie gel stain solution with agitation for 40 minutes and then destain with Coomassie gel destain solution until background staining disappears.
    b) Immunoblotting: If specific protein is needed to be detected by antibody, process gel as described in the protocol of Gel Transfer and Western Blot.

Gel Transfer

A. Reagents and Materials

  • Transfer buffer 10X stock solution: 0.25M Tris, 2M Glycine.  Add methanol to 10% after dilution to 1X buffer and just before use.
  • Blocking buffer: 5.0% non-fat dry milk in 1 X PBS, pH=7.4.
  • PVDF membrane

B. Equipment

  • Electroblotting apparatus: Bio-Rad Trans-Blot Cell
  • Power supply: Bio -Rad PowerPac H

C. Protocol

This protocol is the following steps of SDS-PAGE.

  • PVDF membrane process:  Cut PVDF membrane to the same size of SDS-PAGE gel. Soak membrane sequentially in 100% methanol for 15 seconds, distilled water for 5 seconds and into transfer buffer for at least 10 minutes.
  • Electrotransfer
    a) Arrange gel-membrane sandwich as described in manufacturer’s instruction. Place the transfer sandwich unit into buffer tank, fill with pre-cooled transfer buffer and attach the electrodes. Set the power supply to 100V and transfer for 80 minutes.
  • Blocking
    a) Disconnect transfer apparatus, remove transfer cassette and transfer PVDF membrane to blocking buffer. Rock the blocking membrane on shaker for 2 hours at RT or keep it at 4C overnight.
    b) Follow Immunoblotting protocol as described.

Immunoblotting

A. Reagents and Materials

  • Antibody dilution buffer: 5% non-fat dry milk in 0.1% PBST
  • Developing solution/Fixative solution for X-ray film
  • Membrane washing buffer: 0.1% PBST
  • Primary antibody; Aviva offers >30,000 antibodies for Western Blot
  • Secondary antibody; Goat anti-Rabbit I gG-HRP (CAT#ASP00001)
  • Horseradish Peroxidase Substrate: e.g. Chemiluminescent FemtoMax (CAT# OORA01695)
  • X-ray film

B. Equipment

  • Rotary shaker

C. Protocol

This protocol is to be followed after Gel Transfer.

    • Primary antibodies preparation
      a) The final primary antibody concentration should be 1.25 - 5.0 ug/ml for protein A purified IgG (_T100) and 0.25 - 1.0 ug/ml for peptide purified IgG (_P050). See product details for exact dilution to use.
    • Pretreat membrane: Wash the membrane in wash buffer for 10min.
    • Primary antibodies incubation
    • a) Dilute the primary antibody in antibody dilution buffer to suitable antibody concentration, then incubate for 1hour at room temperature with agitation to enable adequate homogeneous covering of the membrane and prevent uneven binding. Overnight at 4C with agitation is also acceptable.
    • Wash membrane: Pour off primary antibody solution and wash membrane 6 times for 5 minutes each time in Membrane washing buffer.
    • Secondary antibodies incubation
    • a) Dilute secondary antibody in antibody dilution buffer (1:15,000 from 1mg/mL stock) and incubate with shaking for 1 hour.
  • Wash membrane: Pour off primary antibody solution and wash membrane 6 times and for 5 minutes each time in Membrane washing buffer.
  • Development
  • a) Mix the two solutions of HRP substrate reagent in appropriate ratio immediately prior to adding to the membrane. Incubate with the membrane for 1 minute. Drain and remove excess reagent and place the membrane in plastic wrap to ensure a dry surface for film exposure.
  • Exposure in darkroom
    a) Exposure the membranes to X-ray film in cassette for 1 minute. Manual film development is used to control the incubation time of the x-ray film in the developing solution and fixative solution. Normally, about 2-10 min for development and 5 min for fixation. Dry the film after thoroughly washing with water.

 
See these other western blotting resources:

What is a western blot?

    Fred Hutchinson

western blotting

    protocol



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