WesternBlots - ImmunoBlots                             

 

INSTRUCTION FOR USE

Materials required but not provided

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Disposable tip micropipettes to dispense volumes of 10, 20 und 1000 l

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Range of standard, clean volumetric laboratory glassware

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Freshly distilled water

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Whirlmix

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Clean disposable glass or plastic tubes for serum dilution

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Absorbent paper towels

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Shaker

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Tweezers

Serum specimens

Freshly collected sera intended for H. pylori-testing should be stored at 2 8 C before use.

If it is intended to store the specimens for periods in excess of 48 hours they should either be preserved with 0,1 % sodium azide or frozen at 20 C.

Contaminated, haemolysed or excessively lipaemic sera may give aberrant results.

Heat inactivation at 56 C should not affect the test results.

Instruction for reagent preparation and storage

All unopened components can be used up until the date printed on the outer box, provided they are stored at 2 ‑ 8 C.

The strips should be stored at room temperature after first opening.

Reconstitution of the serum-dilution-washing buffer (SDW)

Add 20 ml concentrated washing buffer to 180 ml distilled water (= 200 ml). Mix well. Working strength solution is stable up to two weeks at 2 8 C.

Conjugate

While the first incubation is in progress, dilute the conjugate:

number of strips needed

serum - dilution - washingbuffer (ml)

concentrated conjugate (l)

1

1,0

10

2

2,0

20

3

3,0

30

4

4.0

40

5

5,0

50

etc.

etc.

etc.

It is not necessary to bring the conjugate to room temperature prior to dilution. It is recommended to take the conjugate out of the refrigerator, draw the needed amount from the vial and put the remaining conjugate back into the refrigerator immediately. Working strength solution is stable for one hour at room temperature (22 C).

 ASSAY PROCEDURE

1. Take the required number of strips out of the tube with tweezers and put them the right side up in their respective channels of the incubation tray. The upper side of the strips (with the strip number and the front marker line) must never turn down during all incubations. If that happens while adding reagents or buffers, turn the strip back the right way up immediately with tweezers.

2. Add 2 ml blocking-buffer (mix well prior to use) to each strip. Take care that all strips are completely covered with fluid. That is important for all other steps of the procedure, too.

3. Incubate for 15 min on a shaker. Take care that the fluid is mixed well by the shaking but cannot contaminate the adjacent channel. That is especially important for the following serum incubation!

4. Dilute serum specimens 1 : 51 with SDW buffer (add 20 l specimen to 1.000 l SDW buffer and mix well).

5. Discard the blocking-buffer by decanting from the incubation tray. The strips are completely re-hydrated now and will stay adhesive to the bottom of the channels during decanting. Add 1 ml diluted serum specimen to their respective channels and incubate for 40 minutes on the shaker.

6. Wash all strips 4 times. Sufficient washing is very important:

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Inclining the tray carefully, aspirate the channel contents using a vacuum line fitted with a trap. Avoid cross-contamination!

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Add 1 ml SDW buffer, shake gently for some seconds and aspirate.

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Add 2 ml SDW buffer, shake for 3 minutes and aspirate. Repeat this step two times. For the last two steps, aspirating can be replaced by decanting.

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Invert the tray and tap firmly on absorbent paper towels.

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Check that there is no residual wash-buffer in the channels.

7. Add 1 ml freshly prepared conjugate to each strip.

8. Incubate for 30 minutes on the shaker.

9. Wash all strips 4 times as described in step 6. Aspirating can be replaced by decanting.

10. Wash all strips with distilled water (don't incubate). This extra washing improves the development of the bands and reduces background staining.

11. Add 1 ml substrate to each strip.

12. Incubate for 10 15 minutes on the shaker.

13. Add 1 ml stop solution to each channel, shake gently for some seconds and decant. Add 1 ml stop solution again, shake for 3 minutes and decant.

14. Remove the strips with tweezers from the tray and dry them on absorbent paper. Analyse the band pattern according to the procedure on the next page.

For documentation store the strips protected from light.

summary of the procedure - Algorhythm

 Westernblot
Put the strips into the incubation tray and add 2 ml blocking-buffer

incubate 15 minutes on a shaker and decant

dilute serum specimens 1 : 51 with SDW buffer and mix well
add 1 ml diluted specimen to the strips
incubate 40 minutes on a shaker

rinse strips 4 times

add 1 ml freshly prepared conjugate to all strips

incubate 30 minutes on a shaker

rinse strips 4 times

add 1 ml substrate to all strips
incubate 10 - 15 minutes on a shaker
add 1 ml stop solution to all strips
analyse band pattern with kit-specific template or Westernblot Scanner Software

 

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E-mail: postmaster@westernblot.ru