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Antibody Purification from Western Blotting    

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This protocol describes a method of purifying antibodies from sera with denatured antigens immobilized on western blot membranes. Advantages include (1) fast and easy; (2) purification of antibody with antigen in denatured form allows high yield in case antigen protein solubility is limited. Disadvantage is that possible antibodies that recognize certain 3D structure in solution of antigens might not be purified using such a method. Regarding this issue, the flow through is recommended to be kept and can be used for other purification methods with folded antigens.

Materials and Reagents

  1. Antigen protein
  2. PVDF or Nitrocellulose membrane (Amersham biosciences/GE Healthcare Dharmacon)
  3. Ponceau S (Sigma-Aldrich, catalog number: P7170 )
  4. non-fat Milk powder (Carnation, any your favorite brand in local store)
  5. Tween 20 (Sigma-Aldrich)
  6. Glycine
  7. Acetic acid
  8. 2 M Tris-HCl (pH 8.5)
  9. NaCl
  10. KCl
  11. Na2HPO4
  12. KH2PO4
  13. Ultra pure water
  14. TBST (see Recipes)
  15. 10x TBS (1 L) (see Recipes)
  16. PBS (pH 7.4) (see Recipes)
  17. Ponceau S solution (see Recipes)


  1. Dialysis bag (Thermo Fisher Scientific/Pierce Antibodies, catalog number: 68035 )
  2. Comb


  1. Load 1-10 mg purified antigen protein into appropriate SDS-PAGE gel.
    Note 1: High amount of antigen protein is essential to retrieve antibody in high purity and yield. To load as much protein into the gel as possible:
    1. Use wide comb (vendors normally have comb with only two wells: one well is in regular size for marker, the other well takes the rest of the space).
    2. Insoluble protein can be purified under denaturing conditions.
    3. Even for soluble protein, higher yield can be achieved by eluting directly from the purification beads by SDS loading buffer.
    Note 2: If your protein has a big tag such as GST, MBP, NusA, you should always run a preclearance of antibody against these tags from the serum with blot containing these tag proteins.
  2. Transfer to PVDF or Nitrocellulose membrane (note, if your protein size is small, choose 0.2 μm pore size rather than 0.45 μm).
  3. Stain the membrane with Ponceau S solution and cut the strip containing your antigen protein as small as possible.
  4. Pre-elute the membrane with 100 mM glycine (pH 2.5) for 10 min. Rinse with TBST.
  5. Block with 5% non-fat milk in TBST for 1 h at room temperature (RT).
  6. Rinse with TBST three times.
  7. Incubate the 5-10 ml antiserum overnight at 4 °C. Depending on the membrane size.
  8. Wash with TBST three times.
  9. Elute the antibody by incubating the membrane with 1-2 ml 100 mM Glycine (pH 2.5) for 2 min.
  10. While incubating, add 75 μl-100 μl 2 M Tris-HCl (pH 8.5) to tubes. The volume should allow a final concentration of 150 μM Tris after step 11.
  11. After 2 min, add 1-2 ml of the eluted antibody in glycine from step 9 to the tubes from step 10. The Tris (pH 8.5) should neutralize the acidic glycine.
  12. Repeat step 9-11 for three times.
  13. Dialyze the fractions against PBS (pH 7.4) and determine antibody concentration as your regular protein work.
  14. Test the antibody with western, immunoprecipitation and immunofluorescence.


  1. 10x TBS (1 L)
    24.23 g Tris
    80.06 g NaCl
    Mix in 800 ml water
    pH to 7.6 with HCl
    Adjust volume to 1 L.
  2. TBST (1 L)
    100 ml of 10x TBS
    900 ml ultra pure water
    500 μl Tween 20
    Note: Tween 20 is very viscous and will stick to the tip of your measuring pipettes. Be sure you take the right amount and add in buffer by pipetting up and down. At the same time, the buffer should be stirring.
  3. PBS (pH 7.4) (1 L)
    8 g of NaCl
    0.2 g of KCl
    1.44 g of Na2HPO4
    0.24 g of KH2PO4
    800 ml H2O
    Adjust pH to 7.4.
    Adjust volume to 1 L with additional distilled H2O.
    Sterilize by autoclaving
  4. Ponceau S solution (1 L)
    1 g Ponceau S
    50 ml Acetic acid
    Add H2O to 1 L


This protocol was adapted from Kurien (2009) and developed in Guowei Fang’s lab, Departmental of Biological Sciences, Stanford University, CA, USA. Funding from the NIH supported this work.


  1. Kurien, B. T. (2009). Affinity purification of autoantibodies from an antigen strip excised from a nitrocellulose protein blot. Methods Mol Biol 536: 201-211.
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Fang, L. (2012). Antibody Purification from Western Blotting. Bio-protocol 2(6): e133. DOI: 10.21769/BioProtoc.133.

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Ming-Chieh Lin
National Taiwan University
The protocol is very easy to follow and my antibody works well after affinity purification, thanks for sharing. My question is, after the elution step, the antigen strips can reuse right? how many times can the strips be re-binded with serum and repeat the elution steps? I kept the serum flow through after first binding (9mL serum+2 strips) and I want to capture rest antibodies retain in the serum flow through. Thank you.
5/26/2017 1:17:43 AM Reply
Yuanqing Lin
I have problms with the elution of the antibodies. I checked with a second antibody if those from the serum are still on the menbrane and they are still there. All of them. I don't get a signal on WB with the eluate, and BCA neither shows higher concentration of protein in comparison to elution buffer with Tris. I variated the elution temperature (0- 25 °C) but I am still gtting the same result...
Do you have a solution for my problem?
7/12/2011 5:16:57 PM Reply

"BCA neither shows higher concentration of protein in comparison to [elution buffer with Tris]. "

Did you elute with Glycine or Tris? Tris will not elute the protein at all. If you used Glycine, have you checked the pH which is critical for the elution.

8/31/2011 4:01:38 PM

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