FAQ
Voltage rises more than 10 V during the transfer when I’m running a semidry transfer unit. Why and how should I proceed?
The stack may be drying which can damage the sample and the transfer unit. Another indicator of buffer depletion is increasing temperature. Please feel the top of the cover periodically for excessive heat. For this reason transfers should not exceed one 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.
Voltage rises and Current drops when I’m running a semidry transfer unit . Why?
The voltage rise and the current drop is a natural observation in semidry transfer (or in any application where you run electricity through a buffer solution). A voltage rise and current drop is the result of buffer depletion.
Can I blot Ettan DALT gels?
The Ettan DALTtwelve and DALTsix do not have a Blotting Kit. The DALT II is not designed or marketed for the purpose of blotting. It is meant to be a component in the Ettan line, followed by a Spot Picker and a Mass Spec.
If you need to blot an Ettan DALT gel, the only stand-alone blotting unit that can accommodate a gel of that size is the TE77 SemiPhor semi-dry transfer unit. To keep the high throughput, you would have to purchase 5-10 of these units, and the power supplies to connect them too. For precast gels, first remove the GelBond backing with a Multiphor II Film Remover.
Hints and tips on transferring proteins by the semi-dry technique.
- Run the transfer as soon as possible after electrophoresis to avoid protein diffusion within the gel.
- If layering several gel sandwiches, transfer gels of only the same size during any run.
- Limit transfers to one hour or less.
- Recommended methanol concentration for different membrane types:
Membrane type | Methanol % |
Charged nylon | 0 |
Nitrocellulose | < 20 |
PVDF | < 15 |
- Use a buffer with low ionic strength such as the two listed below to prevent overheating. Use the alternate CAPS buffer when Tris cannot be used, as in peptide sequencing. CAPS can improve transfer because of its effect on the charge of the protein (See Matsudaira, P. (1987) J. Biol Chem 262:10035).
Towbin buffer
(25 mM Tris, 192 mM glycine, 20% v/v methanol, pH 8.3, 1 liter)
Tris (FW 121.1) 25 mM 3.0 g
Glycine (FW 75.07) 192 mM 14.4 g
SDS* (FW 288.4) 0.1 % (3.5 mM) 1.0 g
Dissolve in 600 ml distilled water. Add methanol as required**.
Bring to 1 liter with distilled water. Do not adjust the pH, which should be between 8.2 and 8.4.
Optional: Chill before use.
*Optional: Adding SDS can improve transfer efficiency.
**Depending on the membrane type selected, adding methanol can improve the transfer results (see discussion and table above). Because buffers containing methanol may deteriorate if stored for long periods, add methanol as required just prior to transfer.
CAPS buffer, 1X
(10 mM CAPS, pH 11.0, 1 liter)
CAPS (FW 221.3) 10 mM 2.2 g
[3-(cyclohexylamino)-l-propanesulfonic acid]
Dissolve in 600 ml distilled water, adjust to pH 11.0 with conc. HCI. Adjust volume to 1.0 liter.
- For a 3-buffer system, refer to Kyhse-Anderson, J. (1984) J Biochem and Biophys Meth 10:203-209.
- Transfer efficiency varies depending on the gel concentration, which can be optimized. See Hoefer Technical Bulletin 141 or Smejkal and Gallagher (1994) Biotechniques. Feb 16, #2, 196-202
Can I pause overnight at any step in the silver staining process?
At all points following the fixing step, the procedure must be carried through to completion, but the fixing step can be prolonged for several days.
Tips to improve transfer efficiency
1. Check that the buffer pH is close to the intended pH. Most buffers should not be titrated; make fresh buffer.
2. Use 3.5 mM SDS (0.1%) in the transfer buffer.
3. Use reagent-grade chemicals.
4. Increase the net charge on the protein by using 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.
5. If using a non-nitrocellulose membrane, avoid including methanol in the transfer buffer or reduce the amount to the minimum possible.
Can I blot a GelBond-backed gel?
Electrotransfer cannot occur while the GelBond is stuck to one side of the gel. Remove the GelBond backing (with the film remover)and the gel can be blotted using any conventional transfer unit.
Consumables
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | PlusOne Glycine | 17132301 | 71.57 USD |
Add to cart
|
2 | Tris | 17132101 | 120.41 USD |
Add to cart
|
3 | Sodium Dodecyl Sulfate | 17131301 | 75.15 USD |
Add to cart
|
# | Product Name | Product Code | Price | |
---|---|---|---|---|
Amersham ECL Western Blotting Detection Reagent | 25006262 | 347.00 USD |
Add to cart
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Amersham ECL Western Blotting Detection Reagent | 25006265 | 157.68 USD |
Add to cart
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Amersham ECL Western Blotting Detection Reagent | 25006327 | 250.00 USD |
Add to cart
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Amersham ECL Western Blotting Detection Reagent | 25020024 | 451.00 USD |
Add to cart
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Amersham ECL Prime Western Blotting Detection Reagent | 28980926 | 345.00 USD |
Add to cart
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Amersham™ ECL Select™ Western Blotting Detection Reagent | 29013864 | 420.00 USD |
Add to cart
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Amersham ECL Prime Western Blotting Detection Reagent | 29018618 | 705.00 USD |
Add to cart
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Amersham ECL start Western Blotting Detection Reagent | 29117182 | 173.95 USD |
Add to cart
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|
Amersham ECL start Western Blotting Detection Reagent | 29117183 | 278.00 USD |
Add to cart
|
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | Sodium Dodecyl Sulfate | 17131301 | 75.15 USD |
Add to cart
|
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | Sodium Dodecyl Sulfate | 17131301 | 75.15 USD |
Add to cart
|
Spare parts
# | Product Name | Product Code | Price | |
---|---|---|---|---|
3 | Blotting paper | 80621129 | 94.46 USD |
Add to cart
|
7 | Adaptor kit (4 mm male / 4 mm female) | 80610527 | 50.52 USD |
Add to cart
|
Troubleshooting
Find solutions to product related issues. For unlisted issues please contact local Cytiva service representation.
Blots in the same stock transfer unevenly
Possible cause | Suggested remedy |
---|---|
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. |
No current flows through the stack
Possible cause | Suggested remedy |
---|---|
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
Possible cause | Suggested remedy |
---|---|
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. |
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. |
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. |
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. |
Voltage rises more than 10 V during the transfer
Possible cause | Suggested remedy |
---|---|
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. |
Guide to Transfer Format: Tank vs Semi-dry Transfer
Tank Transfer | Semi-dry Transfer | |
---|---|---|
Flexibility | High or low current. Fast or slow transfer. Recirculating coolant and/or magnetic stirring. |
Limited to rapid transfer with minimal buffer without cooling. |
High Molecular Weight Proteins | Efficient transfer. | Variable efficiencies. |
Quantitative vs. Qualitative Results | Quantitative transfer of low molecular weight proteins under conditions that allow efficient binding to the membrane. | Some "blow through" of small molecular weight proteins and qualitative transfer of large molecular weight proteins. |
Buffer Required | 4-5 L for Transphor transfer units and 1 L for Mighty Small Transphor transfer unit. | 100-250 ml, or just enough to construct a bubble-free sandwich. |
Current Required (for 1-2 h transfer) | 1-1.5 A | Maximum 0.8 mA/cm2 current and maximum 1 hour transfer time. Stack can be re-wetted with fresh transfer buffer and transferred for an additional 1 hour (repeat as required). |
Temperature Control | Optional Heat Exchanger for refrigerated water recirculation provides transfers as low as 10 °C in TE42 and TE52X units. | Temperature regulation is not possible and therefore currents and transfer times must be limited (see above). |
Gel Capacity | TE62X Transphor transfer unit holds up to 4 standard gels with cooling, TE52X Transphor unit holds up to 2 standard gels with cooling, TE22 Mighty Small Transphor holds up to 4 mini-gels with cooling. | TE70 SemiPhor transfer unit holds 2 mini-gels or 1 standard gel without stacking, TE77 SemiPhor transfer unit holds 4 mini-gels, 2 standard gels or 1 IsoDALT gel without stacking. |
Nucleic Acid Transfers | Possible. | Not recommended. |
The bands appear smeared or diffuse on the membrane
Possible cause | Suggested remedy |
---|---|
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. |
Uneven band transfer to the membrane. Regions of the membrane show bands that are more intense than in other regions
Possible cause | Suggested remedy |
---|---|
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. |
Hints and tips on transferring proteins by the semi-dry technique
• Run the transfer as soon as possible after electrophoresis to avoid protein diffusion within the gel.
• If layering several gel sandwiches, transfer gels of only the same size during any run.
• Limit transfers to one hour or less.
• Recommended methanol concentration for different membrane types:
Membrane type | Methanol % |
Charged nylon | 0 |
Nitrocellulose | 20 or less |
PVDF | 15 or less |
• Use a buffer with low ionic strength such as the two listed below to prevent overheating. Use the alternate CAPS buffer when Tris cannot be used, as in peptide sequencing. CAPS can improve transfer because of its effect on the charge of the protein (See Matsudaira, P. (1987) J. Biol Chem 262:10035).
Towbin buffer
(25 mM Tris, 192 mM glycine, 20% v/v methanol, pH 8.3, 1 liter)
Tris (FW 121.1) | 25 mM | 3.0 g |
Glycine (FW 75.07) | 192 mM | 14.4 g |
SDS* (FW 288.4) | 0.1 % (3.5 mM) | 1.0 g |
Dissolve in 600 ml distilled water. Add methanol as required**.
Bring to 1 liter with distilled water. Do not adjust the pH, which should be between 8.2 and 8.4.
Optional: Chill before use.
*Optional: Adding SDS can improve transfer efficiency.
**Depending on the membrane type selected, adding methanol can improve the transfer results (see discussion and table above). Because buffers containing methanol may deteriorate if stored for long periods, add methanol as required just prior to transfer.
CAPS buffer, 1X
(10 mM CAPS, pH 11.0, 1 liter)
CAPS (FW 221.3) | 10 mM | 2.2 g |
[3-(cyclohexylamino)-l-propanesulfonic acid]
Dissolve in 600 ml distilled water, adjust to pH 11.0 with conc. HCI. Adjust volume to 1.0 liter.