干法蛋白转膜

1. Prepare the transfer buffer.

2. Following electrophoresis, equilibrate the gels in transfer buffer. Equilibration facilitates the removal of electrophoresis buffer salts and detergents. The length of time required for equilibration is dependent on the gel thickness. For example, 15 min. for a 0.75 mm SDS-PAGE gel.

3. Cut the membrane to the dimensions of the gel. Wet the membrane by slowly sliding it at a 45° angle into transfer buffer and allowing it to soak for 15-30 min.

4. Cut filter paper to the dimensions of the gel. Two pieces of thick filter paper (or six pieces of thin filter paper) per gel are needed for each gel/membrane sandwich. Completely saturate the filter paper by soaking in transfer buffer.

5. If more than one full-size gel is to be transferred at one time, cut a piece of dialysis membrane with the appropriate molecular weight cutoff to the dimensions of the gel. Completely wet the dialysis membrane in transfer buffer.

Assembly of the Unit for Standard Transfers
(Wear gloves for this procedure to avoid contamination of membranes.)

1. Remove the safety cover and the stainless steel cathode assembly.

2. Place the pre-soaked sheet of filter paper onto the platinum anode. Roll a pipet or test tube over the surface of the filter paper (like a rolling pin) to exclude all air bubbles. If thin filter paper is used, repeat with two more sheets of buffer-soaked filter paper.

3. Place the pre-wetted blotting media on top of the filter paper. Roll out all air bubbles.

4. Carefully place the equilibrated gel on top of the transfer membrane, aligning the gel on the center of the membrane. Transfer will be incomplete if any portion of the gel is outside the blotting media. Roll out all air bubbles.

5. Place another sheet of pre-soaked filter paper on top of the gel, carefully removing air bubbles from between the gel and filter paper. If thin filter paper is used, place three sheets on top of the gel, and remove bubbles from between each layer.

6. If more than one full-size gel is to be transferred, place sheet of pre-soaked dialysis membrane on top of the filter paper stack. Repeat the procedure from step 2. Up to four mini gels can be transferred at the same time by placing them side-by-side on the anode platform.

7. Carefully place the cathode onto the stack. Press to engage the latches with the guide posts without disturbing the filter paper stack.

8. Place the safety cover on the unit. Plug the unit into the power supply. Normal transfer polarity is cathode to anode, i.e., red wire to the red outlet and black wire to black outlet on the power supply.

Caution: Do not reverse polarity. This will result in damage to the stainless steel cathode.

9. Turn on the power supply. Transfer mini gels for 15-30 min. at 10-15 V. Large gels can be transferred for 30 min. to 1 hr. at 15-25 V. Do not exceed 25 V with this instrument. A current limit (3 mA/cm2 for large gels; 5.5 mA/cm2 for mini gels) is recommended to prevent excessive heating during the run. Under the strong fields developed by this apparatus, transfers may not always be quantitative. A certain quantity of protein may be transferred through the membrane and onto the filter paper below. (i.e. Constant current ~ 15 QmM for 2 mini gels)

10. Following transfer, turn the power supply off, and disconnect the unit from the power supply. Remove the safety cover and the cathode assembly. Discard the filter paper (and dialysis membrane, if used). The transfer efficiency can be monitored by staining the gel with Coomassie blue R-250 protein stain or with Bio-Rads Silver Stain Kit. Alternatively, prestained molecular weight standards can be used, or a portion of the membrane can be stained for total protein with colloidal gold, Biotin Blot Total Protein Stain, or an anionic dye such as Amido Black. Zeta-Probe membrane can be stained with the Biotin-Blot Total Protein Stain.

SDS may be added to Buffer 1 to increase protein elution from the gel:

48mM Tris, 39 mM glycine, (20% methanol), 1.3 mM SDS (0.0375%), pH 9.2.
Dissolve 5.82 g Tris and 2.93 g glycine, and 0.0375 g SDS or 3.75 ml of 10% SDS in ddH2O (add 200 ml of methanol); adjust the volume to 1 liter with dilute ddH2O.

DO NOT ADD ACID OR BASE TO ADJUST pH.

Towbin transfer buffer for SDS-proteins using nitrocellulose (with methanol) or Zeta-Probe membrane (without methanol):

25 mM Tris, 192 mM glycine (20% methanol), pH 8.3
Dissolve 3.03 g Tris and 14.4 g glycine in ddH2O (add 200 ml of methanol); adjust volume to 1 liter with ddH2O.

DO NOT ADD ACID OR BASE TO ADJUST pH.