Notes

1. We perform electro-transfer from gels to nitrocellulose membrane, using a wet blotter or tank apparatus, where the gel is submerged in a large volume of buffer during the transfer. For two dimensional or large gels, we use a Bio-Rad transfer cell with 3 L of transfer buffer or an ISODALT cell (Hoefer Scientific Instruments) with 20 L of transfer buffer. ISODALT cells allow the simultaneously transfer of 5 gels. For minigels we use Mini Trans-Blot cell (7.5 x 10 cm blotting area). The transfer buffer can be used several times, if stored at 4°C. "Semi-dry" electro blotters require smaller volumes of buffer, since only the membrane and filter paper have to be wet and the procedure is faster. However, the "wet" method is recommended when antigen is present in small quantities (such as low abundance spots in 2D gels) and/or its molecular weight is high (31). It offers more options, such as temperature, time and voltage control.

2. The transfer buffer we use was first described by Towbin et al. (1). Methanol is toxic and it can be omitted (32-34). Still, we use it to reduce swelling of the gel during transfer and to increase the binding of proteins to nitrocellulose (2,32,35). When working with high molecular weight proteins, elimination of the methanol results in a significant increase in protein transfer efficiency. Some recipes recommend the addition of low concentration of SDS to the buffer to help the transfer of high molecular weight proteins (30) and to improve the transfer of a variety of proteins (32). However SDS reduces the amount of protein bound to the membrane (2) and may adversely affect immunoreactivity by inhibiting renaturation of antigenic sites (36).

For semi-dry blotting it is possible to use discontinuous buffer and/or "elution promoting" buffer on the gel side and a "retention promoting" buffer on the membrane side (37).

3. Reagent grade methanol must be used because trace impurities in methanol can increase the conductivity of transfer buffer and decrease transfer efficiency.

4. PVDF may also be used (38,39). Remember that unlike nitrocellulose, PVDF is a hydrophobic membrane and it must to be presoaked in methanol before use with aqueous solution. The buffer generally used to transfer proteins to PVDF is 10 ml 3-[cyclohexylamino]-1-propanesulfonic acid, 10% (v/v) methanol, pH 11.0 (40), although it is possible to use the buffer described in Towbin et al. (1).

5. Electrotransfer is usually conducted immediately after the electro-phoretic run from unstained gels. However, transfer of proteins from polyacrylamide gels after Coomassie blue or silver staining, has also been reported (41-43). Proteins can also be transferred for immunodetection from gels previously stained in a reverse (negative) way, for example with imidazole-zinc salts (44). In these procedures, immunoreactivity patterns on the membrane, and total protein patterns can be obtained from the same gel from which spots have been transblotted, facilitating matching even with poorly reproducible 2D separations.

6. Polyacrylamide IEF gels should be pre-equilibrated with transfer buffer containing 1% SDS and 20% glycerol, instead of methanol (to prevent swelling), since the focused proteins are at their isoelectric point and do not transfer well without equilibration. If it is desired to maintain the proteins in their native conformation, the SDS may be omitted, but the pH of the transfer buffer should be increase to pH 8.8 and the transfer may be less efficient (45).

7. The transfer efficiency is adversely affected by high molecular weight and the basic pis of some proteins. Therefore, while attempting to transfer these slow proteins it is possible that some faster proteins cross the nitrocellulose membrane and are lost. In cases like this, one can use two stacked membranes, or membranes with smaller pore diameter, which will prevent the loss of small polypeptides during membrane manipulation (46,47). See ref. 48 for information on blotting on various membranes.

Some low molecular weight, basic proteins, such as histones, lysozymes, cytochromes, and so forth, do not transfer well because they may be near their pI in currently used buffers, as SDS is lost during the transfer in methanol. Transfer of these proteins can be improved, without impairing transfer of other proteins, by introducing a more basic transfer buffer and/or omitting the equilibration (Subheading 3.1., step 3 [49]). Alternative buffers have also been proposed (50).

8. When the transfer is conducted at high voltage it is necessary to refrigerate the transfer tank with a thermostatic circulator.

9. If it is only necessary to check the immunoreactivity of an antibody towards a mixture of antigens, without attributing it to a particular protein, the antigen mixture (2-5 ^L) can be spotted directly on the membrane. This technique, known as dot blotting (51) is useful for fast screenings of many antibodies simultaneously, for example in production of monoclonal antibodies.

10. In SDS gels, multicolored proteins can be used to provide a visual display of marker proteins on the transfer membrane. Various companies sell precolored standards and there is in the literature (52) a procedure for generating multicolored molecular weight proteins using a variety of Remazol-reactive textile dyes.

11. Chemical staining of protein patterns transblotted onto the nitrocellulose or other membrane plays an important role in 2D immuno-affinity identification, since it provides "landmark" spots to match immunoreactivity patterns to silver-staining patterns (see Subheading 3.5. and Note 18). Several staining procedures can be chosen. This step is usually carried out before the incubation of transblotted membranes with antibodies, using dyes (e.g., Ponceau S, Fast Green, Amido black) or metal-chelates, which do not interfere with protein immunoreactivity (46,53-56). Staining with substances such as Ponceau S, Fast Green, and metal-chelates is reversible, eliminating interference in the immunoreactivity pattern obtained with chromogenic substrates, but it is not very sensitive. A dye-based staining method, using Direct Blue 71 was recently developed. It is reversible, compatible with immunodetection and with a sensitivity that is 10-fold higher than Ponceau S (57). Permanent staining can also be used if the immunoreactivity pattern is collected from ECL-impressed films, but the stain must not interfere with immuno-reaction.

Fluorescent dyes have also been recently introduced for membrane staining. For example, SYPRO Ruby protein blot stain is a new, luminescent metal chelate stain composed of ruthenium in an organic complex that interacts non-covalently with proteins. This stain is more sensitive than Ponceau, Coomassie blue, Amido Black or India Ink and is nearly as sensitive as colloidal gold staining. This fluorescent stain is fully compatible with immunoblotting (58). When radioactive labeling is possible, the more accurate total protein patterns can be collected from transblotted membranes by phosphor-imaging. The two images will have the same dimensions so that general alignment and recognition of immunoreactive spots in the total protein pattern can be easily achieved. However the requirements for safe handling of radioactive proteins is a huge limitation to this method.

Perfect alignment of immunoreactive spots to total protein pattern can be obtained, at least with PVDF membranes, by the conjunction of colloidal gold staining for total protein detection and ECL for im-munoreactivity on the same membrane (59). This procedure produce an ECL-impressed film with low exposure allowing the detection of immunoreactive spots and an ECL-impressed film with a strong exposure that produces a background pattern. The final result is a single image where immunoreactive spots appears as dark black spots and the general protein pattern appears as light grey spots. Colloidal gold (60,61) and India Ink staining (62) can be applied also after immunodetection. The latter approach is possible if membrane blocking is carried out using Tween-20 only (63,64). Finally another method has been proposed by Zeindl-Eberhart (65) to localize easily imaged antigen on 2D gels. Proteins are transferred to PVDF membrane, immunostained with specific antibodies using Fast Red or 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium as a detection system, and then counterstained with Coomassie brilliant blue. The membrane appears with immuno-stained spots colored in red or black and the total protein pattern in blue. The blocking proteins are removed during the staining with Coomassie and so do not create background staining. In all these methods proteins are transferred onto one membrane and both total protein staining and immunostaining are performed on the same membrane. "Double-replica" blotting methods have also been developed to obtain a membrane with all the proteins stained and that is an almost identical copy of the immunostained one. The first of its kind was described by Johansson (66) who found that by chang ing the direction of the blotting current the proteins could be transferred simultaneously from one gel onto two membranes, on either sides of the gel. A second method described by Neumann and Mullner (67) combines the usual electroblotting procedure with the generation of a "contact copy" from a gel. Both systems enable one membrane to be immunostained whereas the second membrane is stained using highly sensitive total protein staining methods. Protein identification is then carried out by comparing the signals from both matrices.

Similarly, a fast and simple method to produce print-quality like Ponceau replicas from blots was recently described (68). The positive replicas are the same size as blots and can be stored without loss of intensity. This makes them useful for localizing immunoreactive spots in complex 2D electrophoretograms.

12. For a membrane from a 1D SDS-PAGE or IEF gel, we use 3-5 mL of solution depending on the dimension of the membrane in each washing and incubation step. For a 16- x 18-cm membrane, we use 50 mL of solution. In general, volumes can be proportionally adjusted to other membrane dimension. It is important that the membrane is entirely soaked in solution during washing and incubation step.

13. Our blocking procedure is suitable for routine use. However, special conditions and reagents are required for immunoblotting with some antibodies, such as anti-phosphotyrosine antibodies (20,69,70). Information on different blocking conditions can be found in references (51,71-73). Chemicon have developed a new blocking agent, composed of non animal proteins, that ensures uniform blocking, without non specific-binding, eliminating all crossreactivity between animal antigens and primary and secondary antibodies. Blocking with a non-ionic detergent such as Tween-20, without added protein has also been used with the advantage that after immunodetection the blot can be stained for total protein pattern (63,74,75) (see Note 10). On the other hand, it has been found that blocking with detergent alone may cause loss of transblotted proteins (75,76). Using PVDF membrane it is possible to use a nonblock technique: this method incorporates three cycles of methanol-water hydration of the membrane, allowing multiple erasure and probing of the same blot with little or no loss of signal (77).

A modified Western blotting protocol has been developed that increases the binding specificity of antigens and antibodies without increasing the background. The method is based on intermittent microwave irradiation of the blotting membrane during the immunoblotting step, using 5% skim milk as the diluting buffer (78). A simple method to improve Western blotting and reduce background has been developed by Wu et al. (79), that consists of a few modifications to the washing steps and buffer conditions.

14. Fixation of proteins to the membrane can be used to prevent their elution during washing and incubation steps. Some methods have been tried (35,80), but epitopes are sensitive to this treatment and may no longer be detectable by antibody. Another method is to fix the antibody-antigen complexes to the nitrocellulose membrane with glutaraldehyde, after they had been formed (81).

15. Optimal dilution of the primary and secondary antibody should be determined by immunoblotting of one-dimensional gels. Dot blot analysis can also be used. Working solutions of antibodies can be stored at -20°C and used several times (82).

16. Secondary antibodies often give problems of crossreactivity, especially when being used to analyze samples containing antibodies (such as immunoprecipitates, immune tissue, plasma), even when antibodies from different species are used. Langstein and Schwarz proposed a method to avoid this problem, that consists of preconjugating the primary and secondary antibodies (83). Another solution to this problem is "double-blotting." After the membrane has been incubated with the primary antibody, it is blotted a second time under acidic conditions. Antigen and interfering proteins remain bound to the first membrane, and the primary antibodies are transferred to the second one, which can be probed with secondary antibodies, without non specific binding (84).

17. The secondary antibody we use is labeled with peroxidase. The major drawback of this approach is that the range of protein loading that can be used to give a linear relationship between the amount of target protein and the signal is quite limited. Considerable advantages for quantitative analysis can be gained by the use of a secondary antibody coupled to fluorophores that allow quantification of fluorescent signal, e.g., by means of a phosphor imager device. This approach theoretically gives a linear signal through a broad range of protein loading (85,86).

18. ECL detects horseradish peroxidase-conjugated antibodies through oxidation of luminol, in the presence of hydrogen peroxide and a phenolic enhancer under alkaline conditions. ECL reagents are capable of detecting 1-10 pg of protein antigen. An alternative enhancer that extends the duration of light emission is ECL plus (Amersham Biosciences, ref. 87). These systems are suitable for the use of charge-coupled device cameras that require longer exposure times for good quantification of immunoreactions.

19. If chemiluminescence is too strong or background is too high, one can change the detection system to a chromogenic substrate. We use 4-chloro1-naphthol (88) as a chromogenic substrate, according to the following protocol.

a. After ECL detection (or after step 7 of Subheading 3.3.1.), wash the membrane briefly with Tris-HCl 0.05 M, pH 6.8.

b. Soak it in developing solution 20 mL of Tris-HCl 0.05 M, pH 6.8, 7 pL of H2O2 30% (v/v); and 5 mL of 4-chloro1-naphthol 0.3% (w/v) in methanol) until the color appears. Stop the reaction by washing in distilled water.

c. Air dry the membrane and photograph it as soon as possible, because the color fades with time.

20. Chemiluminescent probes enable highly sensitive quantitative analysis of proteins blotted from electrophoretic gels onto a supporting matrix. For a quantitative comparison, it is important to be able to correct for introduced variables such as antibody titre, temperature, substrate etc. Comparison of blots completed on different days requires a chemiluminescent standard. The situation is more complex with 2D gels, where only one sample per gel/blot is used. A method has been published for preparing a chemiluminescent standard for quantitative comparison of 2D Western blot (89).

21. It is also possible to perform stripping with kits as the CHEMICON Re-Blot™ Western blot recycling kit. Stripping of antibodies also elutes antigens from the membrane and signal intensity decreases in successive cycles. It is important therefore to remember that stripping should be used only for qualitative purpose. As alternatives to stripping, one can use:

a. Alternative chromogenic substrates for peroxidase at each cycle (rainbow blotting, [82]).

b. ECL followed by inactivation of peroxidase after each cycle (82).

c. Different labels and detection methods at each cycle (90).

22. To perform the matching process we use the software Melanie 4 (Gene Bio).

23. Matching can also be conducted by inspection by eye of the nitrocellulose and ECL film when the sample contains relatively few spots, all of them detectable by chemical staining of nitrocellulose. In the majority of cases samples are very complex and many low abundance proteins occur. In these cases matching by computer is necessary, in order to identify immunoreactive spots in silver-stained patterns. The following manual procedure is suggested:

a. Match the exposed film with the nitrocellulose membrane, aligning the upper left corner and the two corresponding borders and placing the cut lower right corner in the same orientation for both.

b. Using a waterproof pen, mark the other two borders of the nitrocellulose on the film and transfer the chemically stained spot present on nitrocellulose on the ECL film in order to use them as landmarks for the next matching with the silver nitrate stained gel.

c. Nitrocellulose membrane and film maintain the initial size, but the size of the gel increases after silver staining. Size equalization can be obtained by photographic or photocopy procedures.

d. On a transilluminator match all the landmarks with the corresponding spots on the silver nitrate stained gel to identify the im-munoreactive spots.

When the area of the membrane containing the protein of interest is known, another procedure, described by Lindahl (91), can be used. Only a limited area of the nitrocellulose containing the proteins is cut out and incubated with antibodies. The rest of the membrane is stained with one of the methods described in Note 10 (a method not compatible with immunodetection, but much more sensitive than Ponceau S, can be used). Thus the protein spots can be located in the 2D electrophoresis pattern, matched with a corresponding silver stained gel and translated into the protein pattern. This method also allows a considerable saving of antibodies.

24. We use a computing densitometer 300 S from Molecular Dynamics with a resolution of 4000 x 5000 pixels, 12 bits/pixel, which generates 40 megabytes images on 16 bits.

25. The silver stained image used for matching can be taken from your file archive or from images available on the Internet, provided that identical electrophoretic procedures have been applied. The possibility of matching images derived from different 2D electrophoretic procedures has been investigated by Lemkin P. (92).

26. This step may be difficult if the "landmark" spots stained by Red Ponceau on the nitrocellulose membrane are few. To aid recognition

Fig. 1. Mapping of immunoreactive spots on 2-D silver-stained reference image.

A: Blot membrane stained with Ponceau-S digitized image acquired, before immunodetection, with Image Master VDS-CL system (Amer-sham Biosciences).

B: Digitized image of silver-stained gel. Size is equalized to that of image A on the basis of anchors (arrows).

A' Digitized image of impressed ECL film on blot membrane reported in A.

Recognition of anchor positions (arrows) between A and B and of immunoreactive spots between A and A' allows localization of immuno-reactive spots on silver-stained gel.

Fig. 1. Mapping of immunoreactive spots on 2-D silver-stained reference image.

A: Blot membrane stained with Ponceau-S digitized image acquired, before immunodetection, with Image Master VDS-CL system (Amer-sham Biosciences).

B: Digitized image of silver-stained gel. Size is equalized to that of image A on the basis of anchors (arrows).

A' Digitized image of impressed ECL film on blot membrane reported in A.

Recognition of anchor positions (arrows) between A and B and of immunoreactive spots between A and A' allows localization of immuno-reactive spots on silver-stained gel.

of the spots chosen as landmarks on the silver stained gel, we suggest also staining the gel from which the proteins were transferred with silver. The amount of protein loaded onto this gel must be twice that used for a normal silver-stained gel. Most spots will still be visible on the transferred gel and can be used for an initial matching with the membrane. Using the gel from which the membrane was obtained, the landmarks can be localized correctly. The landmarks are then easily transferred to the silver stained gel by computer matching.

27. The mapping of immunoreactive spots on 2D silver-stained reference images can be improved if the images of the Ponceau and the immuno-stained membranes are scanned using the same system, enabling detection of the total protein pattern directly on to the membrane where the immunoreaction took place. In commercially available gel and blot scanning systems, images generated in different ways can be readily acquired on the same instrument.

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