Procedures

Preparations

• Stock of secondary antibody: Resolve pellet of secondary antibody (CyTM5-conjugated AffiniPure Goat Anti-Human IgG + IgM + IgA (H + L)) into 1.5 ml Aqua dest. till you have a clear solution (check with pasteur pipette; spin down if necessary) and add 1.5 ml of glycerol to get a total antibody concentration of 0.75 mg/ml. Store 50 µl-alequots at -20 °C.

• PBST buffer (PBS buffer containing TweenR 20): Mix BupHTM Phosphate Buffered Saline Packs with Aqua dest. (500 ml per package) and stirr well. Add 0.5 ml TweenR 20 to the 500 ml, use a truncated 1 ml-pipette tip for this viscous fluid. Take care to bring TweenR 20 into solution completely.

• Dilution of primary and secondary antibodies: Dilute blood sera (containing the primary antibodies) and an alequote of the secondary antibody to your designated final concentration using PBST buffer.

Only use freshly prepared PBST buffer and recently diluted antibodies!

Experiments

• Incubation with primary antibody:  Drop 350 µl of diluted primary antibody on the RepliTopes Peptide Array Slide respectively. Take care not to touch the slides with pipette tips and avoid air bubbles! Place the coverslips carefully to get a liquid layer without bubbles. Incubate slides for 2 hours at room temperature, protect them from light. Whensoever you wash the slides, use 200 ml of fresh PBST. Each slide has to be washed 4 times and every washing step should consist in not too rapid movements of the slides during 5 minutes. The coverslips have to be removed carefully by moving the slide up and down in a vertically orientation in the first batch of buffer. Avoid cross-contamination of serum-antibodies by using seperate washing containers for each slide.

• Incubation with secondary antibody (attention: light-sensitive!): Drop 350 µl of diluted secondary antibody on the RepliTopes Peptide Array Slide respectively. Take care not to touch the slides with pipette tips and avoid air bubbles! Place the coverslips carefully to get a liquid layer without bubbles. Incubate slides for 2 hours at room temperature, protect them from light. The first 3 slide washings have to be done in 200 ml of fresh PBST every time, for 2 more washings use 200 ml of fresh Aqua dest. both times. Move the slides to clean racks before you start with the water-washes. Every washing step should consist in not too rapid movements of the slides during 5 minutes. The coverslips have to be removed carefully by moving the slide up and down in a vertically orientation in the first batch of buffer. This time, up to 4 slides can be washed in one container because all slides have been incubated with the same antibodies.

Immediately (!) after the second water-wash, centrifuge slides for 5 minutes at 700 rpm to dry them completely.

Store slides in dark and dry environment till the scan and also afterwards!

Material