WesternBlottingProtocol

實驗概要

The western blot  (sometimes called the protein immunoblot) is a widely used analytical  technique used to detect specific proteins in the given sample of tissue  homogenate or extract. It uses gel electrophoresis to separate native  proteins by 3-D structure or denatured proteins by the length of the  polypeptide. The proteins are then transferred to a membrane (typically  nitrocellulose or PVDF), where they are probed (detected) using  antibodies specific to the target protein.

主要試劑

1X Cell Lysis Buffer

5X SDS sample Buffer

Transfer Buffer

10X TBS-T (Tris-bufferedsaline containing Tween-20)

Blocking Buffer

Wash Buffer

Primary and Secondary Antibody Dilution Buffer

Alternate Blocking Buffer

Alternate Primary and Secondary Antibody Dilution Buffer

Blotting Membrane

10X SDS Running Buffer

實驗步驟

1. Sample preparation:

1)         Place cells in a micro-centrifuge tube and centrifuge to collect cell pellet.

2)         Lyse the cell pellet with 100 μl lysis buffer on ice for 30min (For 1X 106 cells, lyse with 100 μl lysis buffer).

3)         Centrifuge at 14,000 rpm (16,000 x g) for 10 min at 4 °C.

4)          Transfer the supernatant to a new tube and discard the pellet. Remove  20μl supernatant and mix with 20 μl of 2x sample buffer.

5)         Boil for 5min. Cool at RT for 5min. Micro centrifuge for 5min.

6)         Load up 40μl of sample to each well of a 1.5mm thick gel*.

7)         Set gel running conditions according to the manufacturer’s instructions.

8)          Transfer the proteins to a nitro cellulose or PVDF membrane with  variable power settings according to the manufacturer’s instructions.

     *Guidelines for choosing gel percentages are based on protein size to  be detected: 4-5% gel, >200 kD; 7.5% gel, 120-200 kD; 8-10% gel,  40-120 kD; 13% gel, 15-40 kD; 15% gel, <20kD.

2. Membrane Blocking:

1)          Remove the blotted membrane from the transfer apparatus and  immediately place in blocking buffer consisting of 5% nonfat dry  milk/TBS-T**.

2)         Incubate the blot for 1 hr at room temperature, or overnight at 4 °C with agitation.

3. Antibody Incubation:

1)          Dilute the primary antibody to the recommended concentration/dilution  in 5% nonfat dry milk/TBS-T** (usually at 1 μg/ml). Place the membrane  in the primary antibody solution and incubate 2 hrs at room temperature,  or overnight at 4 °C with agitation.

2)         Wash three times for 5 min each with Wash buffer (TBS containing 0.1% Tween-20).

3)          Incubate the membrane for 30 min at room temperature with horse radish  peroxidase (HRP)-conjugated secondary antibody, diluted to 1:1000 in  5%  nonfat dry milk/TBS-T**.

4)         Wash 4 times for 10 min each with TBS containing 0.1% Tween-20, and once for 2 min with PBS.

4. Protein Detection:

1)          Incubate membrane (protein side up) with 10 ml ECL (enhanced  chemiluminescence substrate) for 1-2min. The final volume required is  0.125 ml/cm².

2)         Drain off excess detection reagent, wrap up the blot sand gently smooth out any air bubbles.

3)          Place the wrapped blots, protein side up, in an X-ray film cassette  and expose to x-ray film. Exposure scan vary from 5 seconds to 60 min.

注意事項

High background

1)     Transfer buffers may have become contaminated. Contamination can be  transferred to the blots from electrophoresis and related equipment used  in blot preparation.

2)     Post-antibody washes may not have been performed for a sufficient  period of time or were not performed in a high enough volume.

3)    The blocking and incubation agents used were not freshly prepared or were too dilute.
 

No signal or poor signal

1)   Transfer efficiency may have been poor. Check protein transfer by staining the gel and/or membrane.

2)    Incorrect storage of antibodies or ECL western blotting detection reagents may result in a loss of signal.

3)     Insufficient protein may have been loaded on the gel. Depending on the  location of the target protein, membrane or nuclear preparations may be  required (instead of whole cell lysates).

4)    Film exposure time may have been too short.