Western blot | Antibody incubation & Washing

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Lab. Bioclock & Aging/Dewan Md. Sumsuzzman

Antibody incubation & Washing

※ When you first take the Antibody, it tells you how much concentration should be diluted. Use it at that concentration. If the protein band comes out, continue to use it. If the band does not come out, increase the concentration.

Primary & secondary antibody incubation

※ If there are several proteins you want to see, the membrane may be cut to size. Prior to Primary Ab treatment, remove the water, wrap it in a wrap, and check for protein markers (total volume 2-3 ml).

A. Dilution of primary Ab:

Dilute primary Ab in TBST (0.5% Skim milk / TBST) and incubate shaking incubation for 1 hour at room temperature. Or overnight at 4 ° C.

→ 0.5% skim milk + TBST + primary Ab

→ Used primary Ab can be re-used, can be kept at 4 ℃

Ex 1) TBST 10 ml + Skim milk 0.05 (g) + 10 μl of primary AB when the dilution concentration is 1: 1000 and the total volume is 10 ml

Ex 2) TBST 2 ml + Skim milk 0.01 (g) + 2 ㎕ of primary AB when the dilution concentration is 1: 1000 and the total volume is 2 ml.

➢ The membrane is washed three times for 10 minutes with TBST buffer

→ If the membrane is turned upside down, it will not wash properly.

→ If the membrane is cut, use 3-5 ml

B. Dilution of Secondary AB:

dilute the secondary AB in TBST (or 1% skim milk / TBST), then shake the membrane at room temperature for 1 hour.

→ TBST + primary Ab

Ex 1) When the dilution concentration is 1: 1000 and the total volume is 10 ml, TBST 10 ml + 10 ㎕ of secondary Ab

Ex 2) When the dilution concentration is 1: 1000 and total volume is 2 ml, TBST 2 ml + 2nd Ab 2 ul.

2.Washing:

➢ The membrane wash 5 times with TBST solution every 10 minutes.

→ If the membrane is inverted, the washing will not work properly

C. Washing:

➢ The membrane wash 5 times with TBST solution every 10 minutes.

→ If the membrane is inverted, the washing will not work properly.

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