Western blot

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Schematic structure of a Western blot chamber with marked anode and cathode.

Western blot ( Western blot ) refers to transmission (engl. Blotting ) of proteins on a carrier membrane , which can then be detected by different reactions. The transfer can be done in different ways: by diffusion , capillary action or electrophoresis . The Western Blot is used in biochemical and medical research as well as in diagnostics , making it one of the most widely used protein analytical methods. The western blot belongs to the group of immunoblots .

history

The Western Blot method was originally developed in 1979 in the Robert Nowinski laboratory at the Fred Hutchinson Cancer Research Center in Seattle by W. Neal Burnette and independently in the laboratory of George R. Stark at Stanford University . In the same year Harry Towbin and co-workers were able to change the procedure to nitrocellulose as in the simpler Southern Blot , which is the simpler and preferred method nowadays.

The name of the blot method ("Western Blot") comes from the English blot for blob or stain and from English. blotting paper for blotting paper , which also creates an identical imprint of the original. This was first introduced in 1981 by Neal Burnette as an allusion to Northern Blot and Southern Blot (the publication appeared two years late because the journal had originally rejected it). Edwin Southern is considered to be the inventor of the blotting technique. In 1975 he developed a method for the separation of DNA fragments and subsequent hybridization , which he called Southern Blot . The corresponding separation of RNA fragments was referred to as Northern blot based on its name . This is why protein blotting with SDS was called Western Blot . For studying protein-protein interactions has been far-western blotting developed. As a combination of the Western and the Southern blot, the Southwestern blot was developed for the detection of DNA-protein interactions , as was the Northwestern blot for the detection of RNA-protein interactions.

There is no Eastern blot per se . However, the term "Eastern Blot" is used for various methods, e.g. B. for an electrophoretic separation and a transfer of the proteins to membranes with a cationic detergent (e.g. CTAB or 16-BAC), in which the proteins migrate in the opposite direction (towards the cathode). The term Eastern Blot has also been used for blotting lipids on membranes ( Far Eastern Blot ), transferring native proteins from non-denaturing gels, or blotting molecules.

principle

Before the actual Western Blot, a protein mixture is separated using a gel electrophoresis technique in a carrier matrix ( SDS-PAGE , native PAGE , isoelectric focusing , 2D gel electrophoresis , etc.) according to its size, charge or other properties. The proteins to be examined are first separated into protein bands by gel electrophoresis (usually a polyacrylamide gel with an optimal acrylamide concentration).

Protein transfer

Semi-dry blotter for electric transfer
Tank blotter for electrical transfer

In the Western Blot, an electric field directed perpendicular to the polyacrylamide gel is usually applied (electrotransfer) , whereby the SDS-loaded and negatively charged proteins migrate towards the anode (plus pole). If there is no time pressure, the transfer can alternatively take place by capillary action in the direction of a dry stack of a hydrophilic , adsorbent material (capillary transfer) or by diffusion .

During the transfer, the proteins migrate from the gel onto a membrane, e.g. B. nitrocellulose , nylon , fiberglass or mostly polyvinylidene difluoride ( PVDF ). PVDF membranes are first briefly placed in methanol so that the hydrophobicity of the membrane is reduced and the transfer buffer can come into contact with the PVDF membrane. With nylon or PVDF, proteins stick to the membrane surface due to hydrophobic and polar interactions, while adsorption with nitrocellulose or glass fibers takes place via ionic and polar interactions.

For an electrotransfer, the membrane is placed on the anode side of the gel and covered on both sides with filter papers wetted with transfer buffer and placed between the two electrodes . Three different systems are used for electrotransfer: the tank blot system, the semi-dry blot system and the dry blot system, which differ in terms of structure and buffer quantities and systems used. In electrotransfer, an electric current of 2.5  mA / cm² of the blot membrane is mostly used; H. with a blot membrane of 10 cm × 10 cm size, 250 mA are set in the electrophoresis power supply .

With capillary transfer, on the other hand, the gel is placed on a transfer buffer-wetted filter paper, on which in turn the membrane and finally a dry filter paper stack is placed.

The pattern of the electrophoretic separation is retained during the transfer. The proteins can now be used for other methods (e.g. binding of an immunoconjugate ). After this process, the SDS attached to the proteins can be washed out. As a result, the proteins can renature and partially take up their secondary and tertiary structure again, but due to the spatial separation of the various subunits of a protein, the quaternary structure can not be restored in this way. To determine the number and size of the subunits of a protein, however, covalent crosslinking of the subunits can be carried out prior to SDS-PAGE , which survives boiling in sample buffer for SDS-PAGE and can reveal the structure of a protein complex after immunostaining .

Protein detection

Blot membrane after protein transfer and ponce staining of all proteins. A pre-stained protein marker is separated in the left lane.
Structural formula of Amido Black, the sodium salt of which Amido Black 10 B is used as a dye

The temporary staining of all proteins on the blot membrane allows the protein transfer to be checked and the amount of protein (charge control) in the various traces of the gel to be assessed prior to an immunodetection. Originally individual were for this purpose household proteins such as Tubulins or actins made visible, however, the staining of all proteins advantages. The entirety of the membrane-bound proteins can be made visible via certain dyes. Examples are Ponceau S , colloidal gold , amido black or Indian ink . Other dyes are capable of post-translational modifications such as B. to mark phosphorylated proteins. The stains frequently used in SDS-PAGE, such as Coomassie staining or silver staining, allow only a slight renaturation of the proteins during the decolorization prior to an immunodetection and also incompletely decolorize. Fluorescent colors have a larger linear dynamic range in two-dimensional applications and allow a more precise quantity determination, such as B. the total protein staining with trichloroethanol or epicocconone . Depending on the experimental setup, individual proteins can also be selectively made visible using other methods, e.g. B. radioactively labeled antibodies or other proteins that are radioactively labeled by the incorporation of isotopes during protein synthesis or by subsequent phosphorylation , in the case of enzymes by converting an appropriate substrate .

If an unstained size marker (synonym: comigration standard ) was used in addition to the proteins to be examined , reversible staining of all proteins on the membrane with z. B. Ponceau S made. The subsequently visible marker bands can be obtained by applying mechanical pressure to the membrane and thus used for protein identification even after the color reaction. Reversible protein staining is not required for pre-stained size markers.

blocking

After the protein transfer, the remaining free sites for protein binding on the membrane are blocked with a protein or chemical polymer that cannot be recognized by the antibodies . As a result, no further, undesired proteins can subsequently adhere to the membrane and produce undesired colorations in the antibody staining. Solutions of defatted milk powder , bovine serum albumin ( BSA , bovine serum albumin), gelatine and other proteins or solutions of polyvinylpyrrolidone with a mild detergent (such as Tween 20 or Nonidet P40 ) are suitable for this .

Immunodetection of individual proteins

Immunodetection scheme. The primary antibody binds to its antigen, which is fixed on the blot membrane. The secondary antibody binds to this in turn for detection
Western blot of a nitrocellulose membrane with a protein in different amounts (from left: 17 ng, 13 ng, 11 ng, 8 ng, 4 ng), the detection was carried out by chemiluminescence (ECL) on X-ray film
Western blot with fluorescence-labeled secondary antibody

The protein bands of individual proteins are mostly identified on the membrane with the help of specific antibodies that bind to individual epitopes in the protein of interest . By placing the protein-laden blot membrane in dilute solutions of specific antibodies ( monoclonal ) or mixtures of specific antibodies ( polyclonal ), the antibodies bind to the appropriate protein band on the membrane. This antibody that binds directly to the protein is called the primary antibody. Unspecifically bound antibodies are removed again by washing steps with buffers (mostly three times for 10 minutes each time with TBS-T buffer ) containing detergents . The affinity of the binding between antigen and antibody is used: an antigen-specific primary antibody only binds to “its” epitope on the antigen, which is spatially separated from the other proteins and has a characteristic molar mass . Because renaturation is often not complete, problems can arise when using monoclonal antibodies that recognize a discontinuous epitope (synonym: conformational epitope) on the protein. With antibodies that can recognize a continuous epitope (synonym: sequence epitope), renaturation is not necessary.

The detection usually takes place with an immunoconjugate consisting of a signal-generating molecule coupled to a further antibody (secondary antibody). A secondary antibody binds to the Fc region of the primary antibody as an antibody conjugate (immunoconjugate) with a reporter enzyme , which is used for detection after further washing steps (usually three times for 10 minutes each time with TBS-T buffer). The use of a secondary antibody conjugate instead of a primary antibody conjugate allows it to be used in a modular fashion for various primary antibodies with associated cost savings, since the coupling of each individual primary antibody with a reporter enzyme is no longer necessary. Furthermore, the secondary antibody increases the signal, since the polyclonal secondary antibody, directed against several epitopes on the Fc fragment of a species , can bind to several locations in the Fc area of ​​all primary antibodies of a species and groups many reporter enzymes there. Reporter enzyme-antibody conjugates (immunoconjugates) are commercially available. In enzyme-coupled immunoconjugates, the enzyme catalyzes a color or chemiluminescence reaction (ECL), e.g. B. with an HRP -coupled secondary antibody. HRP catalyzes the conversion of luminol or other dioxetanes into its oxidized form, the luminescence of which can be detected. The luminescence reaction takes place z. B. in a solution of 100 mM TRIS / HCl pH 6.8; 0.2 mM p -umaric acid (dissolved in dimethyl sulfoxide ); 1.2 mM luminol (sodium salt, dissolved in dimethyl sulfoxide) and 0.01% (v / v) hydrogen peroxide .

Depending on the question, the immune blot can - as described below - the antigen or - such as e.g. B. in the HIV test - the primary antibody can also be the search object. Antibodies are also proteins, and therefore their antigenic properties can also be used, for example, in Western blotting. B. make visible in immunoblot, ELISPOT and ELISA .

Immunoblot vs. (EL) ISA

Both are methods that serve to detect antigens (proteins) with the help of labeled antibodies: Both are ISAs ( I mmuno s orbent A ssay), methods from the field of proteomics .

The immunoblot extends the ELISA to a certain extent by the dimension of electrophoretic separation at the expense of a selective enrichment of the antigens by coating antibodies. This lack of enrichment can be achieved through additional protein purification or immunoprecipitation . Through gel electrophoresis and fixation on a solid medium (the blot membrane), the antibodies are able to "choose" different antigens at different, defined locations. B. check a serum using a large number of blotted antigens for this large number of associated antibodies, but due to the denaturation during sample preparation for SDS-PAGE almost exclusively continuous epitopes (synonymous sequence epitopes) are detected. Due to the spatial separation, several components against which a serum contains antibodies can also be detected in parallel and selectively. This effect can be achieved by sera from immunized or convalescent patients (polyclonal) or by mixtures of monoclonal antibodies.

Applications

The Western blot is one of the most widely used protein analysis methods. It is estimated that at least 8–9% of the work currently published in this field uses Western blots. In the field of protein biochemistry , the Western Blot is used for the qualitative detection of individual proteins and protein changes such as post-translational modification . A semiquantitative analysis can also be performed (sample A contains more protein X than sample B), and by applying a dilution series of a known protein concentration, the estimate of the amount of a single protein on the blot can be compared more precisely. When cloning a vector for the production of recombinant proteins , the Western blot is used to check the protein synthesis of the respective protein after transfection of eukaryotic cells and approximately two days of cell culture or after transformation of bacteria and approximately one day of culture .

In the field of medicine, Western blotting is used to detect diagnostically relevant proteins, for example antibodies in blood serum , which can be typical for the presence of certain infectious diseases , e.g. B. in the HIV test . In addition, this method helps in research in the search for disease-relevant proteins such as B. the BSE pathogen PrPSc or HIV . Various proteins, such as ERK , which occur frequently in tumors , can also be quantified using Western blotting and thus degenerated cells can be recognized. Also z. For example, it can be determined to what extent certain drugs have a regulatory effect on the increased expression of such proteins in the cell and thus have an effectiveness against the further growth of tumor cells.

literature

Web links

Individual evidence

  1. ↑ a b CP. Moritz: 40 years Western blotting: A scientific birthday toast . In: Journal of Proteomics . February 10, 2020. doi : 10.1016 / j.jprot.2019.103575 . PMID 31706026 .
  2. ^ Rajendrani Mukhopadhyay: W. Neal Burnette: the man behind the Western Blot , ASBMB Today 2012.
  3. ↑ Jaime Renart, Jakob Reiser, George E. Stark: Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. In: Proceedings of the National Academy of Sciences . Vol. 76, No. 7, 1979, pp. 3116-3120, PMID 91164 , PMC 383774 (free full text).
  4. ^ Harry Towbin, Theophil Staehelin, Julian Gordon: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. In: Proceedings of the National Academy of Sciences . Vol. 76, No. 9, 1979, pp. 4350-4354, PMID 388439 , PMC 411572 (free full text).
  5. ^ W. Neal Burnette: "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. In: Analytical Biochemistry . Vol. 112, No. 2, 1981, pp. 195-203, PMID 6266278 , doi : 10.1016 / 0003-2697 (81) 90281-5 .
  6. ↑ Citation's Classic: W. Neal Burnette (PDF; 240 kB)
  7. ↑ Engelbert Buxbaum: Cationic electrophoresis and electrotransfer of membrane glycoproteins. In: Analytical Biochemistry. Vol. 314, No. 1, 2003, pp. 70-76, PMID 12633604 , doi : 10.1016 / S0003-2697 (02) 00639-5 .
  8. ↑ Dianne T. Akin, Raymond Shapira, Joseph M. Kinkade Jr .: The determination of molecular weights of biologically active proteins by cetyltrimethylammonium bromide-polyacrylamide gel electrophoresis. In: Analytical Biochemistry. Vol. 145, No. 1, 1985, pp. 170-176, PMID 4003759 , doi : 10.1016 / 0003-2697 (85) 90343-4 .
  9. ↑ Joachim Hartinger, Katinka Stenius, Dagmar Högemann, Reinhard Jahn : 16-BAC / SDS-PAGE: a two-dimensional gel electrophoresis system suitable for the separation of integral membrane proteins. In: Analytical Biochemistry. Vol. 240, No. 1, 1996, pp. 126-133, PMID 8811889 , doi : 10.1006 / abio.1996.0339 .
  10. ^ Dai Ishikawa, Takao Taki: Micro-scale Analysis of Lipids by Far Eastern Blot (TLC Blot). In: Journal of Japan Oil Chemists' Society. 47, 1998, p. 963, doi : 10.5650 / jos1996.47.963 .
  11. ↑ Hiroyuki Tanaka, Noriko Fukuda, Yukihiro Shoyama: Eastern blotting and immunoaffinity concentration using monoclonal antibody for ginseng saponins in the field of traditional Chinese medicines. In: Journal of Agricultural and Food Chemistry . Vol. 55, No. 10, pp. 3783-3787, PMID 17455950 , doi : 10.1021 / jf063457m .
  12. ↑ a b A. Goldman, JA Ursitti, J. Mozdzanowski, DW Speicher: Electroblotting from Polyacrylamide Gels. In: Current protocols in protein science / editorial board, John E. Coligan .. [et al.]. Volume 82, 2015, pp. 10.7.1-10.7.16, doi : 10.1002 / 0471140864.ps1007s82 , PMID 26521711 .
  13. ↑ BT Kurien, RH Scofield: Multiple Immunoblots by Passive Diffusion of Proteins from a Single SDS-PAGE Gel. In: Methods in molecular biology (Clifton, NJ). Volume 1312, 2015, pp. 77-86, doi : 10.1007 / 978-1-4939-2694-7_11 , PMID 26043992 .
  14. ↑ CP. Moritz: Tubulin or not tubulin: Heading toward total protein staining as loading control in western blots . In: Proteomics . September 20, 2017. doi : 10.1002 / pmic.201600189 . PMID 28941183 .
  15. ↑ Isabel Romero-Calvo, Borja Ocon, Patricia Martinez-Moya, Maria D. Suarez et al .: Reversible Ponceau staining as a loading control alternative to actin in Western blots. In: Analytical Biochemistry. Vol. 401, 2010, pp. 318-320, PMID 20206115 .
  16. ↑ Fabrizio Gentile, Ernesto Bali, Giuseppe Pignalosa: Sensitivity and applications of the nondenaturing staining of proteins on polyvinylidene difluoride membranes with Amido black 10B in water followed by destaining in water. In: Analytical Biochemistry. Vol. 245, 1997, pp. 260-262, PMID 9056225
  17. ↑ Charlotte Welinder, Lars Ekblad: Coomassie staining as loading control in Western blot analysis. In: Journal of Proteome Research Vol. 10, 2011, pp. 1416-1419, PMID 21186791 .
  18. ↑ Carol L. Ladner, Jing Yang, Raymond J. Turner, Robert A. Edwards: Visible fluorescent detection of proteins in polyacrylamide gels without staining. In: Analytical Biochemistry Vol. 326, 2004, pp. 13-20, PMID 14769330 .
  19. ↑ Jennifer E. Gilda, Aldrin V. Gomes: Stain-Free total protein staining is a superior loading control to β-actin for Western blots. In: Analytical Biochemistry Vol. 440, 2013, pp. 186-188, PMID 23747530 .
  20. ↑ Christian P. Moritz, Sabrina X. Marz, Ralph Reiss, Thomas Schulenborg, Eckhard Friauf: Epicocconone staining: a powerful loading control for Western blots. In: Proteomics PMID 24339236 .
  21. ↑ John W. Haycock: Polyvinylpyrrolidone as a blocking agent in immunochemical studies. In: Analytical Biochemistry. Vol. 208, No. 2, 1993, pp. 397-399, PMID 8095775 , doi : 10.1006 / abio.1993.1068 .
  22. ↑ Klaus Klarskov, Stephen Naylor: India ink staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis and in conjunction with Western blots for peptide mapping by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry. In: Rapid Communications in Mass Spectrometry. Vol. 16, No. 1, 2002, ISSN  0951-4198 , pp. 35-42, PMID 11754245 , doi : 10.1002 / rcm.522 .