FAQ
Some of my proteins do not transfer out of gel during electroblotting.
- Use a thinner gel.
- Reduce the gel acrylamide concentration.
- Do not use staining or fixing agents on the gel before transfer.
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.
What are the recommended conditions for protein transfer with the TE22?
Typical transfers with Towbin buffer should proceed for 1 hour, at 0.4A, 100V, 10oC coolant temperature. Constant current is recommended. Parameters may need to be adjusted empirically for different samples, and buffers
Consumables
# | Product Name | Product Code | Price | |
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3 | PlusOne Glycine | 17132301 | 71.57 USD |
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4 | Tris | 17132101 | 120.41 USD |
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5 | Sodium Dodecyl Sulfate | 17131301 | 75.15 USD |
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# | Product Name | Product Code | Price | |
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Amersham ECL Western Blotting Detection Reagent | 25006262 | 347.00 USD |
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Amersham ECL Western Blotting Detection Reagent | 25006265 | 157.68 USD |
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Amersham ECL Western Blotting Detection Reagent | 25006327 | 250.00 USD |
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Amersham ECL Western Blotting Detection Reagent | 25020024 | 451.00 USD |
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Amersham ECL Prime Western Blotting Detection Reagent | 28980926 | 345.00 USD |
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Amersham™ ECL Select™ Western Blotting Detection Reagent | 29013864 | 420.00 USD |
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Amersham ECL Prime Western Blotting Detection Reagent | 29018618 | 705.00 USD |
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Amersham ECL start Western Blotting Detection Reagent | 29117182 | 173.95 USD |
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Amersham ECL start Western Blotting Detection Reagent | 29117183 | 278.00 USD |
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# | Product Name | Product Code | Price | |
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1 | Sodium Dodecyl Sulfate | 17131301 | 75.15 USD |
Add to cart
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Accessories
# | Product Name | Product Code | Price | |
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1 | Cooling tubing | 80110656 | 302.00 USD |
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The unit has a 4 mm recessed jack. Please see selection guide to identify if you will require an adaptor.
# | Product Name | Product Code | Price | |
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1 | EPS 301 Power Supply | 18113001 | 1,015.00 USD |
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4 | Mains cable, 120 V | 19244701 | 71.49 USD |
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5 | Mains cable 220 V | 19244801 | 110.80 USD |
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Spare parts
# | Product Name | Product Code | Price | |
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1 | Gel cassette | 80620464 | 251.00 USD |
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2 | Cassette foam sponge | 80620521 | 50.09 USD |
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3 | Cassette foam sponge | 80620502 | 57.27 USD |
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4 | Dracon sponge thickness = 6 mm (0.25 inch) | 80642162 | 35.78 USD |
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5 | Blotting paper 9x10.5 cm | 80620540 | 100.81 USD |
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7 | Adaptor kit (4 mm male / 4 mm female) | 80610527 | 50.52 USD |
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# | Product Name | Product Code | Price | |
---|---|---|---|---|
5 | Banana plug | 80617747 | 70.12 USD |
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Troubleshooting
Find solutions to product related issues. For unlisted issues please contact local Cytiva service representation.
Cassette is bowed, resulting in incomplete or swirled transfers
Possible cause | Suggested remedy |
---|---|
Overpacking the cassette, and then running the unit at temperatures in excess of 50 °C causes the cassettes to become bowed. Bowed cassettes will always pack improperly and cause diffuse or swirly band patterns on the membrane. |
Transfer buffer temperatures must be kept below 50 °C. Cooling requires an external cooling water recirculator, such as the MultiTemp III. |
Plastic parts have become warped
Possible cause | Suggested remedy |
---|---|
Plastic parts have become warped |
1. Circulate only water or 50/50 water/ethylene glycol through the heat exchanger. Never introduce anti-freeze or organic solvent into any part of the instrument. Organic solvents will cause irreparable damage to the unit. |
Power supply output current fluctuates
Possible cause | Suggested remedy |
---|---|
Corrosion of electrode post (on electrode panel) or electrode socket (in lid) |
Clean the corroded parts |
- |
Check the power supply with another electrophoresis unit to see if the output is okay |
Loose electrode post on electrode panel. |
Fasten the electrode post |
Incomplete transfer
Possible cause | Suggested remedy |
---|---|
Blank areas on the membrane |
1. Remove all trapped air pockets in the transfer stack assembly: assemble the stack while it is submerged in transfer buffer, gently press on each sponge as it is added to the stack, and roll a glass pipette or test tube over the membrane and gel to eliminate all air bubbles. |
Grid pattern on membrane |
Add extra sheets of blotting paper to increase the clearance between the cassette panel and the gel. Take care not to overstuff the cassette; the gel should be held firmly and evenly between the sponges, but not so tightly that it is squeezed. |
Molecules do not migrate out of gel |
1. Increase the field strength. |
No current flows through the stack
Possible cause | Suggested remedy |
---|---|
Power supply diode/fuse has blown by high current |
Unit requires servicing |
Bubble is present in stack or above bottom electrode, caused by excessive current |
Lower power supply current limit |
All components were not soaked in transfer buffer |
Soak all components in transfer buffer |
Stack is dry or partially dry |
Re-wet stack |
Safety interlock is defective |
Unit requires servicing |
Diffuse band patterns
Possible cause | Suggested remedy |
---|---|
1. Transfer immediately after electrophoretic separation. If equilibrating before the transfer, shorten or eliminate the equilibration time or move the gel to the cold room during equilibration. |
|
Distorted or swirled transfers due to loose stacks |
Increase the number of sponges in the stack |
Banana plug post on the electrode panel is corroding
Possible cause | Suggested remedy |
---|---|
Post has been exposed to buffers. |
Post must be kept clean and dry to avoid corrosion. If corrosion is observed wipe and dry the area. In extreme cases, the banana plug posts and/or the high-voltage leads may need to be replaced. |
Open circuit or no output current
Possible cause | Suggested remedy |
---|---|
Blown fuse in protection circuit. |
Replace fuse. |
Inefficient binding to membrane
Possible cause | Suggested remedy |
---|---|
Chemical parameters are not optimal. |
1. Fix or crosslink the molecule onto the membrane according to the requirements of the nucleic acid, protein, or membrane type. |
Membrane parameters are not optimal. |
1. Wear gloves when handling membranes. |
Uneven band transfer
Possible cause | Suggested remedy |
---|---|
Blotting paper too large |
The blotting paper and membrane must be the same size as the gel or 1– 2 mm smaller. Larger sizes will provide an electrical path for current to bypass the gel solution. |
Different sizes and/or net charges of proteins |
Different proteins will transfer at different rates depending on size and net charge. |
Stack is over-wetted |
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. Please note: No contact or path should be available that allows current to bypass the gel. |
Trapped air pockets between the gel and the membrane |
Remove trapped air pockets between the gel and the membrane during stack assembly. |
Presence of gas |
Lower the current setting to minimize the amount of gas produced by the electrolysis of buffer. |
Buffer has too high ionic strength and hence generated too much current |
Use buffer with a lower ionic strength. Please note: Do not back-titrate transfer buffers |