| Problem |
Cause |
Solution |
| Poor electrophoretic transfer; bands appear weak on blot (ensure proteins have been transferred by staining both the gel and blot with a total stain. For example, stain the gel with Bio-Safe™ Coomassie or SYPRO Ruby stain, and stain the blot with Ponceau S stain). Alternatively, one could use stain-free technology and LF PVDF membranes |
Power conditions were inadequate or transfer time too short |
- Increase the transfer time (thicker gels require longer transfer times)
- Check the current at the beginning of the run; it may be too low for a particular voltage setting, indicating incorrect buffer composition.
- Use high-intensity blotting
- Use a power supply with a high current limit. If an incorrect power supply is used, it is possible to not reach the set voltage if the current of the power supply is at its maximum limit
- Equilibrate gel in transfer buffer for 10 minutes prior to transfer. (Do not do this if using Trans-Blot® Turbo system)
|
| Power conditions were too high or transfer time too long (proteins may transfer through the membrane and into the filter paper) |
- Shorten transfer time
- Reduce transfer voltage
|
| Transfer buffer was incorrect or prepared incorrectly |
- Prepare fresh transfer buffer (never reuse transfer buffer)
|
| Proteins moved in the wrong direction (the gel/membrane sandwich may have been assembled in the wrong order, the cassette inserted in the tank in the wrong orientation, or polarity of the connections may be incorrect) |
- Check the gel/membrane sandwich assembly
- Check the assembly of the transfer cell
- Check the polarity of the connections to the power supply
|
| The charge-to-mass ratio is incorrect (native transfers) |
- Use a more basic or acidic transfer buffer to increase protein mobility. A protein near its isoelectric point (pI) will transfer poorly (buffer pH should be 2 pH units higher or lower than the pI of the protein of interest for optimal transfer efficiency)
|
| Protein precipitated in the gel |
- Use SDS in the transfer buffer. SDS can increase transfer efficiency but it can also reduce binding efficiency to nitrocellulose and affect reactivity of some proteins with antibodies
- Reduce or eliminate the alcohol in the transfer buffer
|
| The power supply circuit is inoperative or an inappropriate power supply was used |
- Check the fuse
- Make sure the voltage and current output of the power supply match the needs of the blotting instrument
- Check the output capacity of the power supply
|
| The gel percentage was too high (decreasing %T or %C increases gel pore size and increases transfer efficiency) |
- Reduce %T (total monomer) or %C (crosslinker). Using 5%C (with bis-acrylamide as the crosslinker) produces the smallest pore size
|
| Regions of poor protein binding on the blot |
The membrane was not uniformly wet before transfer |
- Ensure that membranes are uniformly wet before transfer
- Because of the hydrophobic nature of PVDF, the membrane must be completely soaked in methanol prior to equilibration in aqueous transfer buffer. A completely wet PVDF membrane has a gray, translucent appearance
|
| Buffer tank not filled to correct level |
- Completely fill transfer tank with buffer. Transfer tank must contain sufficient buffer to entirely cover blot area
|
| Swirls or missing bands; bands appear diffuse on the blot |
Contact between the membrane and the gel was poor; air bubbles or excess buffer remain between the blot and gel |
- Carefully move the roller over the membrane in both directions until air bubbles or excess buffer are removed from between gel and membrane and complete contact is established
- Use thicker filter paper in the gel/membrane sandwich
- Replace the foam pads. Pads compress and degrade with time and will not hold the membrane to the gel
|
| White spots on membrane |
The membrane was not properly wetted or had dried out |
- White spots on the nitrocellulose membrane indicate dry areas where protein will not bind. If wetting does not occur immediately by immersion of the sheet in transfer buffer, heat distilled water until just under the boiling point and soak the membrane until completely wet. Equilibrate in transfer buffer until ready for use
- White spots on the PVDF membrane indicate areas where the membrane was either improperly prewetted or allowed to dry out. Because of the hydrophobic nature of PVDF, the membrane must be prewet in methanol prior to equilibration in aqueous transfer buffer. Once wet, do not allow membrane to dry out. If the membrane dries, rewet in methanol and re-equilibrate in TTBS (this may adversely affect downstream detection processes)
|
| Broad or misshapen bands |
Poor gel electrophoresis |
- Artifacts of electrophoresis may occur as a result of poor gel polymerization, inappropriate running conditions, contaminated buffers, sample overload, etc.
|
| Gel cassette pattern transferred to blot |
Foam pads are contaminated or too thin |
- Clean or replace the foam pads
|
| Excessive amounts of protein were loaded on the gel or too much SDS was used in the transfer buffer. Proteins can pass through the membrane without binding and recirculate through tank blotting systems |
- Reduce the amount of protein on the gel
- Reduce the amount of SDS in the transfer buffer
- Add a second sheet of membrane to bind excess protein
|
| The transfer buffer was contaminated |
- Prepare fresh transfer buffer
|
| Overall poor binding to the membrane |
Methanol in the transfer buffer is restricting elution |
- Reduce the amount of methanol. This may improve transfer efficiency of proteins from the gel but it also may decrease binding to nitrocellulose membranes; 20% methanol is generally optimal for protein binding
|
| SDS in the transfer buffer reduces the binding efficiency of proteins |
- Reduce or eliminate SDS from the transfer buffer
|
| Proteins passed through the membrane. Proteins <15 kD may show decreased binding to 0.45 µm membranes |
- Use PVDF or 0.2 µm nitrocellulose (smaller pore size)
- Decrease the voltage if using the high-intensity option
- Place an additional membrane in the gel sandwich to detect proteins that are being transferred through the membrane
|
