GENERAL REMARKS

1.) The blot device should not be run above 40V

2.) If you test new buffer systems in semi dry blotting, assure that the device does not overheat.

3.) We recommend not to change the polarity of the electrodes.

4.) Clean the electrodes just with aqua dest. and dry with paper.

5.) After first use, the anode electrode shows a glittering "print" of the blot sandwich. This is due to oxidation products of the blot buffer and does not impair proper functioning of the electrode.

The Blot Sandwich

1.) Cut 9 sheets of filter paper exactly in size of the gel to be blotted.

2.) Wet 3 sheets of filter paper separately with Cathode buffer.

3.) Place the first sheet onto the electrode and layer the other two individually thereon (see also figure).

4.) Mark the gel and also the blot sheet (nitrocellulose or some other customary blot sheet). This should ensure the unambigious association of sample tracks in the gel and those blotted onto the sheet.

5.) Place the gel for about one min into cathode buffer. The gel is subsequently layered onto the existing stack of filter paper.

6.) Wet 3 sheets of filter paper and the blot sheet separately with Anode-I buffer. Layer the blot sheet first and then the filter paper individually onto the stack.

7.) Wet 3 sheets of filter paper with Anode-II buffer, layer them onto the stack, and close the cover of the blot device.

8.) Connect the blot device with your power supply. Plus to cover minus to stand

Be aware that the steel electrode is always connected to the cathode (MINUS), otherwise it will corrode!

9.) Switch your power supply to constant current operation and choose a current of 0.8 mA per cm² of gel surface. The power should be 5V-6V (Laemmli SDS gel).

10.) Blot for about 30 min to 60 min at constant current. The absolutely optimal time, however, can only be elucidated by trial.

Protein and immunostaining

1.) Soak your blot sheet for 2-5 min in Ponceau S ready-to-use solution

2.) Destain the weakly stained background in 10% acetic acid

remark

1.) The nitrocellose sheet keeps its size, whereas it shrinks during amido black staining. Shrunk blot sheets are usually not suiteable for reference purposes.

2.) Protein bands, which were Ponceau S stained, may be easily destained (see below). Thus the blot may be subsequently immunostained, the antibody binding, however, may be reduced.

B Removal of Ponceau S from protein bands

1.) Discard the acetic acid and rinse the sheet three times with aqua dest..

2.) Incubate the sheet two times for 15 min in Anode-I buffer.

D Immunostaining

1.) Saturation of unspecific protein binding sites:

incubate the agitating blot for 20 min in TBS-5% FCS-1% BSA at room temperature. The amount of solution required depends on the size of the blot sheet and the inkubation vessel used. In general, you should use plastic or glass bowls which are only slightly larger in size than the blot sheet. Add sufficient solution, so that the sheet agitates freely.

2.) Washing:

Wash the blot three times in TBS, 15 min each

3.) Inkubation with antibodies with specificity for immobilized proteins:

Incubate the blot for several hours at room temperature in antibody fraction (i.e. antiserum, IgG) diluted with TBS-1% FCS.The dilution depends on the quality of the antibody fraction and has to be estimated experimentally. As a rule, the dilution should be not as much as in ELISA assays.

4.) Washing (see step 2)

5.) Inkubation with peroxidase conjugated (PO) antibodies with specificity for antibodies immobilized in step 3:

Dilute PO-antibodies about 1:1000 with TBS-1% FCS and incubate for about 2 hours. Commercial PO-antibodies differ strongly in quality and concentration. You therefore should experimentally estimate the optimal dilution.

6.) Washing (see step 2)

7.) Development:

It is very important that you use a glass bowl at this stage, since, for reasons not known, this strongly reduces the background staining! Incubate the blot in TBS-Chloronaphthol and add 5 µl H₂0₂ per 20 ml solution. The development may last seconds to minutes. Depending on the intensity, the immunostained bands appear blue to almost black. The development is stopped (rinse under tap water), when the backhground becomes stained or when the stained pattern does not change any more. The stain is sensitive to light, so you should store the dried blot in the dark (wrap into Alu-sheet).

References

- PO-antbodies in Western Blotting: Hawkes et al. (1982) Anal. Biochem. 119:142

- alkalic phosphatase conjugated antibodies in Western Blotting: Blake et al. (1983) Anal. Biochem. 136:175

- Enzymatic immunoassay in general: P. Tijssen (1985) in "Practice and theory of enzyme immunoassays", Elsevier Scientific Publ., Amsterdam

Affinity purification of antibodies on nitrocellulose

1.) Blot the protein pattern onto nitrocellulose (NC-sheet).

2.) Stain the protein pattern with Ponceau S (see above).

3.) Cut the band of interest out of the NC-sheet.

4.) Destain the protein band.

5.) Perform the steps 1-4 in "Immunostaining". Alternatively, after having performed the steps 1-2 in "Immunostaining", you may store the dried NC-strips for later affinity purification.

6.) Incubate the NC-strip in a small volume of Dissociation buffer.

7.) Collect the supernatant, rinse the NC-strip with Dissociation buffer and pool both supernatants.

8.) Neutralize the pooled supernatant fraction by adding Tris base (ca. 1/6 vol. of 400 mM Tris base; estimate the exact amount by a trial titration) and 0.0185 vol. 10-times concentrated TBS. Finally, for stabilization, add

concentrated BSA solution to give a concentration of 1 %.

9.) The NC-strip should be washed excessively with Dissociation Buffer followed by neutralization with excess TBS. The dried NC-strip may be stored for further use.

Electro-Transfer of DNA to Nylon Membranes

1. rinse gel in aqua dest. after electrophoresis and then incubate at room temperature (RT)in 0.1N HCl until the marker dye (bromphenol blue) changes to yellow (max. 15 min).

2. 2 x 15 min at RT in 0.5M NaOH/1.0M NaCl (denaturation)

3. 2 x 15 min at RT in 0.5M Tris/1.0M NaCl, pH 7.4 (neutralization)

or

Preparation for electro-transfer in 20xSSC buffer (3M NaCl, 0.3 M Na3-citrate):

2. 2 x 15 min at RT in 0.5M NaOH/1.5M NaCl (denaturation)

3. 2 x 15 min at RT in 0.5M Tris/3.0M NaCl, pH 7.4 (neutralization)

Electro-transfer:

The electro-transfer is carried out by use of Semi Dry Blot PEGASUS.

1. Cut filter paper (17MM Whatman) and nylon membrane (BIODYNE, PALL) in size of the gel.

2. Soak filter papers and nylon membrane in electrode buffer (TBE or 20xSSC, according to pretreatment)

3. Place 6 layers filter paper onto the kathode electrode (stand). Avoid enclosure of air bubbles!

4. Layer gel (front side down) onto filter paper stack.

5. Layer nylon membrane onto the gel. Avoid enclosure of air bubbles.

6. Mark the location of the sample troughs on the membrane.

7. Layer 6 more soaked filter papers onto the stack.

8. Close the cover and connect the blot device with your power supply. Plus to cover, minus to stand

9. Switch your power supply the constant current operation and adjust to 1mA/cm².

10. Blot at constant current for 60 to 120 min.

11. stain gel (10 min) and inspect for residual DNA.

12. Rinse blot membrane briefly with 2xSSC and air-dry nylon membrane on fiter paper.

13. Immobilize DNA on the membrane (15 min, 80C).

14. Store dry at room temperature.

APPENDIX

Blot buffers

cathode buffer : 25 mM Tris base, 40 mM 6-Aminocaproic acid, 20% methanol

Anode-I buffer : 30 mM Tris base, 20% methanol

Anode-II buffer: 300 mMTris base, 20% methanol

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Ponceau S staining

Ponceau S stock solution: 200 mg Ponceau S per 100 ml 3 % Perchloric acid

ready-to-use solution : dilute stock sol. 1:5 with 10% acetic acid

Background destaining :10 %acetic acid

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Amido black staining

staining solution : 0.1 % amido black, 45% methanol, 10% acetic acid

background destaining: 25% methanol, 10% acetic acid

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Immunostaining

TBS: 10 mM Tris-HCl, pH=7.4, 150 mM NaCl

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FCS = foetal calf serum

BSA = bovine serum albumin

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*** CAUTION, chloronaphthol is a hazardous compound ***

Chloronaphthol stock solution: 300 mg 4-Chloro-1-naphthol per 100 ml

methanol (---> 0.3 %)

The above solution is stable for several weeks, when stored in an amber

bottle in a refrigerator.

TBS-chloronaphthol: 20.0 ml TBS0.9 ml 0.3 % 4-Chloro-1-naphthol

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Affinity purification of antibodies on nitrocellulose

Dissociation buffer: 100 mM glycine-HCl, pH=2.5