Western blot | Protein transfer

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Lab. Bioclock & Aging/Dewan Md. Sumsuzzman

Transfer membrane

Preparation:

• 10X & 1X Transfer buffer (4℃)

• Sponge / Filter paper / 3MM paper / Membrane

• Cassette / Blotting chamber

• 10X TBS (pH 7.5) &  1X TBST

• 0.2% Ponceau S

• Coomassie blue

• Blocking buffer (5% skim milk in TBST)

• Staining solution & Destaining solution

• BM purple AP substrate

Transfer protocol:

  1. Cut the membranes to the appropriate size and immerse them in transfer buffer.
  2. For PVDF membrane, immerse in methanol for 5 minutes before transferring to transfer buffer.

Membrane activation: The process by which proteins in the gel make hydrophobic interaction with the membrane. This process is not necessary when using an NC.

  1. Transfer the electrophoresis gel to the transfer buffer, remove the wells, and remove the remaining portion except the sample. → Save membrane.
  2. Place the black portion of the Western blot cassette against the bottom of the container containing the transfer buffer and place it on top of it:

→ Immersed in transfer buffer accordingly, dacron sponge / filter paper (1 sheet) / 3MM paper (2 sheets) / gel / Membrane / 3MM paper (2 sheets) / filter paper (1 sheet)/dacron sponge.

→ The gel of the black part of the cassette is the cathode and the membrane of the white part is the anode.

5. Carefully raise the gel on 3MM paper to avoid air bubbles. At this time, I think about the direction of the transfer. If you move the marker to the left, transfer it later and do not reverse the direction when reading the result. → Push the spreader slightly so that the bubble disappears.

6. Carefully raise the membrane on the gel to prevent air bubbles. Place 2 sheets of 3MM paper, 1 filter paper and dacron sponge on it, and gently push it with a spreader to release the bubble.

7. Carefully fasten the cassette to prevent air bubbles from forming and then tighten the clip so that both cassettes do not fall off.

8. After transferring the cassette to the blotting chamber, insert the 1X transfer buffer so that the membrane in the cassette is sufficiently immersed. Place the electrode so that the membrane is the anode and the gel is the cathode.

9. Fill the Bio-Ice cooling unit with ice to prevent denaturation of protein due to heat generated during transfer.

10. Transfer with 230 mA for 1 ~ 1.5 hr.

→ The larger the desired protein size, the longer the time required for the transfer.

→ Prepare the blocking buffer 10 minutes before the transfer is completed.

11. After transfer, remove the membrane, rinse briefly in TBST, and incubate in a compact racker containing blocking buffer (5% skim milk in TBST) for 1 hour at room temperature.

→ It reduces the background by blocking the unbound portion of the membrane in the membrane.

→ If there is no time, put in blocking buffer and overnight at 4 ℃.

→ 2 gels require blocking buffer 30 ml, skim milk 1.5 g (5%) and BSA (-20 ° C storage) 300 μl (1%)

TBST 30 ml + Skim milk 1.5 g → Vortexing + 1% BSA (300 ul)

12. After the transfer, remove the membrane and immerse it in 0.2% Ponceau S to stain.

→Mark the marker area with a marker pen on the dyed marker area.

13. Coomassie staining of transferred gel is used to analyze the cause of failure.

Sandwich assembly:

 

+ve electrode

Sponge

Filter paper

3MM paper

PVDF membrane

Gel

3MM paper

Filter paper

Sponge

-ve electrode

Location: 197 Inje-ro, Hwalcheon-dong, Gimhae, Gyeongsangnam-do, South Korea

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