TE 70 PWR Semi-Dry Transfer Unit, 14 × 16 cm

Blots in the same stock transfer unevenly The blot nearest the anode transfers faster than the blots higher up in the stack.

Yes, this is true, and is a result of an electrical field being stronger closer to an anode plate. That is why it is preferable to lay gels side-by-side rather than stacking them.

Power supply diode has blown by high current (exceeding 1 A).

Unit requires servicing. In the future, lower power supply current limit, and/or make sure the transfer buffer was not back-titrated. Unit must be run at a current limit of 0.8 mA per sq cm of gel. Do not ever exceed this current limit.

Bubble is present in stack or above bottom electrode, possibly generated during the transfer run. This is caused by excessive current

See remedy above

Stack prepared improperly (not all components were soaked in transfer buffer).

Soak all components in transfer buffer

Stack is dry or partially dry.

Re-wet stack. Unit must be run for no more than 1 hour before the stack requires rewetting. Do not ever exceed this time limit.

Safety interlock is defective.

Unit requires servicing.

Transfer was uncomplete

Stain the gel after blotting to test whether the proteins remain in the gel or have blown through the membrane. If the stained gel reveals protein bands, then go to A. If the stained gel is completely blank, go to B.

A. Proteins did not migrate out of the gel.

1. Stack was constructed upside-down relative to the anode.

2. Check all electrical connections. Check that current is flowing by plugging a multi-meter into the circuit and comparing the current reading with the power supply reading.

3. A proper mask must be used!

4. Check that the buffer pH is close to the intended pH. Most buffers should not be titrated; make fresh buffer.

5. Use 3.5 mM SDS (0.1%) in the transfer buffer.

6. Increase transfer time, adding several more sheets of buffer-saturated blotting paper to each side of the gel sandwich so that more buffer is present during the transfer (ie, to avoid buffer depletion). Large fragments may require up to 2 hours, however, current must be limited to 0.8 mA per sq cm of blot --AND-- the stack must be checked after 1 hour to ensure that the stack has not dried out (i.e., re-wet the stack after 1 hour).

7. Do not use staining or fixing agents on the gel before transfer.

8. Use a thinner gel.

9. Reduce the gel acrylamide concentration.

10. If using a non-nitrocellulose membrane, avoid including methanol in the transfer buffer or reduce the amount to the minimum possible.

11. Use reagent-grade chemicals.

12. Increase the net charge on the protein by changing to a transfer buffer with a different pH. Lower pH (<6-7) increases the positive charge on proteins; higher pH (>6-7) increases the negative charge on proteins.

13. Keep in mind that proteins at their isoelectric point will tend to precipitate in the gel. If the isoelectric point of the particular protein of interest is near pH 8.3, try a transfer buffer with a lower pH.

14. High MW proteins migrate slowly (if at all) out of the gel. There is a protease-facilitated transfer method which improves transfer of high MW proteins. See:
Gibson, W, and Benko, D.M. in "CRC Handbook of Immunoblotting of Proteins, Vol 1" pp 93-99, CRC Press, Boca Raton, Florida (1988)
Gibson, W. (1981). Analytical Biochemistry 118, 1.

B.

Stain the membrane (before blocking) with a general stain (eg, amido black). If the membrane shows some bands, but patches where nothing has transferred, go to C. If the membrane is completely blank, then go to D. Note that if the membrane is only Western stained then the problem might lie in the Western staining process.

C. Spots on the membrane where nothing has transferred.

1. Remove trapped air pockets between the gel and the membrane during stack assembly.

2. Lower the current setting to minimize the amount of gas produced by the electrolysis of buffer.

3. Too much current was generated by the use of a buffer with excessive conductivity. Use buffer with a lower ionic strength; do not back-titrate transfer buffers!

4. Are the electrodes flat and firmly attached to the base or top? (Place the flat edge of a plastic ruler on the surface to determine if the center is bulging up. See "Electrodes Are Bulging" section for more details.)

D. Inefficient binding to the membrane. Problems stemming from chemical parameters.

1. Fix or crosslink the molecule onto membrane according to the requirements of the nucleic acid, protein or membrane type.

2. Prepare transfer buffer without SDS.

3. Add 20% methanol to the transfer buffer to enhance binding to nitrocellulose. Add 15% methanol to the transfer buffer to enhance binding to PVDF.

4. If user has not stained the membrane prior to blocking with amido black, then the possibility exists that protein is leaching off the membrane during unnecessarily long incubations in the Western staining.

D. Inefficient binding to the membrane. Problems stemming from membrane parameters.

1. General "blow through" of proteins. Proteins largely bind to membranes electrostatically, so there is an "off rate" for each protein. Reduce transfer time or current settings to optimize the transfer. Place a 2nd membrane behind the 1st membrane to capture proteins that have blown through.

2. Place a membrane on both sides of the gel if you suspect one protein is moving in the opposite direction from the majority of the proteins. Check the top membrane for stray protein(s).

3. Check if too much sample is available for the binding surface area by using two membranes instead of one. If "blow through" occurs, reduce the sample load.

4. Wear gloves when handling membranes.

5. Store membranes at ambient temperature out of direct sunlight to keep the membranes activated. Old membranes stored in a humid environment will give poorer binding.

The stack may be drying out, which can damage the sample and the transfer unit.

Transfers should not exceed 1 hour. If longer transfers are required, turn off the power supply and rewet the stack with fresh transfer buffer. Transfers can then proceed for up to one additional hour.

An indicator of buffer depletion is increasing temperature.

It is advised that the user feel the top of the cover periodically for excessive heat. For this reason transfers should not exceed 1 hour. If longer transfers are required, turn off the power supply and rewet the stack with fresh transfer buffer. Transfers can then proceed for up to one additional hour.

The bands appear smeared or diffuse on the membrane

1. If equilibrating the gel before transfer, shorten or eliminate the equilibration time or move the gel to the cold room during equilibration.

2. If transfer buffer contains methanol (10% or higher), equilibrate the gel in transfer buffer for 30 minutes to allow it to shrink before assembling the stack. NOTE: High molecular weight proteins may not migrate as readily once the pore size is slightly reduced.

3. Take care that the gel does not shift once it contacts the membrane.

4. If excessive heating occurs during the transfer, check that the stack is not drying out, then either shorten transfer time or perform the transfer in a cold room.

5. Check that the preferred binding surface of the membrane (if any) contacts the gel.

6. Check the flatness of the electrodes with the edge of a ruler. If warped, customer must replace. This is not covered by warranty, since the warping occurs only if the unit is used beyond recommended specs (ie, maximum 0.8 mA/cm² of gel; 1 hour blot time). See "Electrodes Are Bulging" section for more details.

Uneven band transfer to the membrane. Regions of the membrane show bands that are more intense than in other regions.

1. Realize that transfers are not quantitative. Different proteins migrate out of gels at different rates (due to size and charge). Different proteins blow through membranes at different rates (mostly due to charge). Thus, each protein will have its own unique optimum transfer condition, and even under the most ideal conditions transfer will never be 100%. If comparing blots of 2 or more different proteins it may be impossible to make quantitative comparisons.

2. Take care to cut the blotting paper and membrane to the size of the gel or 1-2 mm smaller than the gel. No contact or path should be available that allows current to bypass the gel.

3. Do not over-wet the stacks. Buffer should not be oozing out of the stack when the top assembly is placed on it, nor should there be pools of buffer on the bottom electrode. Again, no contact or path should be available that allows current to bypass the gel.

4. Remove trapped air pockets between the gel and the membrane during stack assembly.

5. Lower the current setting to minimize the amount of gas produced by the electrolysis of buffer.

6. Too much current was generated by the use of a buffer with excessive conductivity. Use buffer with a lower ionic strength; do not back-titrate transfer buffers!

7. Are the electrodes flat and firmly attached to the base or top? (Place the flat edge of a plastic ruler on the surface to determine if the center is bulging up. See "Electrodes Are Bulging" section for more details.)