常见问题
I’m running miniVE for 1-D electrophoresis. Do you recommend alumina or glass plates?
The use of alumina plates is not recommended for 2 reasons:
- The gels are surrounded by buffer and therefore both plates should be of the same thermal qualities, else the bands will migrate in "Venetian blind" fashion.
- Alumina plates do not have a clean edge because they are cut by lasers on one side, then tapped to complete the cut--one side is therefore clean, but the other side is scalloped. This presents a greater chance for an imperfect seal against the bottom gasket, and a greater possibility for leakage of the acrylamide solution.
Why is the electrophoresis run on the vertical gel unit running unusually slow or fast?
There are several possible causes for an unusually slow or fast run on a vertical electrophoresis unit:
1. Current leakage around gel. Check for leaks, all plates and spacers must be aligned and free of grease and cracks. If using SE600, make sure that buffer dam is used and assembled properly.
2. Sample or reagent preparation.
a. Check recipes, gel concentrations, and buffer dilution. (For instances, do not use Tris-HCl instead of Tris for Laemmli tank buffer.)
b. If proteins move too fast, dilute the buffer so that less current will be carried.
c. If proteins move too slow, increase the buffer concentration so that more current will be carried.
d. If the required pH of a solution is overshot, do not back-titrate. Discard and prepare fresh buffer.
e. Dispose of older acrylamide solutions and use only stock of the highest quality.
f. Only use fresh deionized urea.
g. Decrease the salt concentration of samples.
3. Voltage or current settings. To increase or decrease the migration rate, adjust the voltage or current by 25 - 50%.
Can I store cast gels?
To store unused gels add approximately 5.0 ml of 1X separating gel buffer to the top of each sandwich, seal with plastic wrap (or heat-seal into a bag), and lay flat in refrigerator set to 4 °C. Or, lay gels flat in a Pyrex baking dish, submerge in 1X separating buffer, and store refrigerated. Use within 1 week.
What sample volumes can I load onto my Mighty Small / miniVE electrophoresis equipment?
Load the sample by underlaying into wells using a fine-tipped microsyringe. The width of the wells depends on the number of wells per comb. If the comb has fewer wells, they are wider, and require more volume to raise the level 1 mm, as shown below:
Number of wells | Comb thickness | Tooth width | Volume of sample/1 mm depth (µl) |
5 | 0.75 | 13.0 | 9.5 |
5 | 1.00 | 13.0 | 12.7 |
5 | 1.50 | 13.0 | 19.1 |
9 | 1.00 | 5.8 | 5.8 |
10 | 0.75 | 4.8 | 3.6 |
10 | 1.00 | 4.8 | 4.8 |
10 | 1.50 | 4.8 | 7.2 |
15 | 0.75 | 2.9 | 2.2 |
15 | 1.00 | 2.9 | 2.9 |
15 | 1.50 | 2.9 | 4.4 |
18 | 1.00 | 2.9 | 2.9 |
The comb teeth are 1.6 cm long.
The recommended amount of protein sample added to each well depends on both the sensitivity of the staining method and the distribution of protein among separate bands. With Coomassie Blue, it is possible to detect as little as 1 µg in a single band. With more sensitive silver stains, it is possible to detect about 10 ng.
Spare parts
Consumables
Accessories
The instrument has a 4 mm recessed jack. Please see selection guide to see if you will require an adaptor.
故障排查
寻找产品相关问题的解决方案。对于未列出的问题,请联系当地的Cytiva服务代表。
Bromophenol blue doesn't sharpen into a concentrated zone in the stacking gel.
可能原因 | 建议的补救措施。 |
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The pH of the stacking gel or running buffer is incorrect |
Check buffer stocks and casting recipe. |
Too high sodium or potassium concentration. |
Avoid using solutions with a high sodium or potassium concentration |
Issues with the acrylamide solution |
|
- |
For best results, allow a stacking gel height of 2.5 times the height of the sample in the well |
Stained sample collected near the top of the gel when the buffer front has reached the bottom
可能原因 | 建议的补救措施。 |
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The gel pore size is too small |
Decrease the % T (Total acrylamide concentration) of the resolving gel |
The protein has precipitated or aggregated |
Heat the sample at a lower temperature (70 °C or less) for 1–2 minutes |
Sample bands are found near top of gel when the buffer front has reached the bottom
可能原因 | 建议的补救措施。 |
---|---|
Gel pore size is too small |
Decrease the %T (Total acrylamide concentration) of the resolving gel |
Protein precipitates or DNA becomes denatured |
Decrease the temperature at which the sample is prepared to 70 °C or less, and limit exposure to heat to 1-2 minutes. |
Protein streaks vertically
可能原因 | 建议的补救措施。 |
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Sample contains particulates |
Centrifuge or filter the sample before loading to remove particulates |
Sample contains salt |
Dialyze or desalt the sample |
Too much sample |
Load less sample |
Proteolytic degradation of sample |
Add protease inhibitors such as PMSF (Phenylmethyl-sulfonyl fluoride) if necessary to prevent proteolytic degradation of sample |
Bands are skewed or distorted
可能原因 | 建议的补救措施。 |
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Sample preparation has to be checked |
Check sample preparation: |
Gel preparation and polymerization have to be checked |
Check gel preparation and polymerization: |
Oxygen is present |
1. Degas the stacking gel solution and avoid trapping air bubbles under the comb teeth. |
Sample contains particulates |
Centrifuge or filter the sample before loading to remove particulates |
Sample contains salt |
Dialyze or desalt the sample |
Bands at buffer front
可能原因 | 建议的补救措施。 |
---|---|
Protein or nucleic acid is not sufficiently restricted by the resolving gel |
Increase the %T (Total acrylamide concentration) |
Incomplete transfer / Blank areas on the membrane
可能原因 | 建议的补救措施。 |
---|---|
Air bubbles in the transfer stack |
Remove all trapped air bubbles in the transfer stack |
Stack is too loose |
Make sure that the stack is not loose. A loose stack will result in smeared bands, or blank areas. Add more sponges to make the stack sufficiently tight. |
Dacron sponges are getting compressed |
1. Over time, the Dacron sponge will get compressed and needs to be replaced |
- |
Use a lower ionic strength buffer |
- |
Check electrode continuity |
Incomplete transfer / Molecules do not migrate out of gel
可能原因 | 建议的补救措施。 |
---|---|
- |
Increase the transfer period. (Try doubling it. CAUTION: the Towbin buffer contained within a blot module contains sufficient ionic strength for transfer up to 2 hours. Do not exceed this length of time or the buffer will become depleted |
Over heated buffer |
Make sure the temperature of the buffer in the blot module does not become overheated. As temperature of the transfer buffer increases, so does conductivity. Higher conductivity means ever higher temperature and you are soon in a positive feedback loop where the buffer is brought to a very high temperature. Should the temperature be high enough, you will simply be "cooking" your proteins in your gel--denaturing them and making them difficult to migrate out. Use passive cooling (fill the tank with ice water) and monitor the temperature periodically during transfer. |
Gel has been exposed to staining or fixing agents before transfer |
Do not expose the gel to staining or fixing agents before transfer. |
- |
Try one or several of the following suggestions: |
Presence of methanol |
Methanol shrinks polyacrylamide gels, making it more difficult for proteins to exit. Avoid including methanol in the protein transfer buffer, or reduce the amount to the absolute minimum. |
The tracking dye starts reversing direction, traveling to the cathode instead of the anode
可能原因 | 建议的补救措施。 |
---|---|
If this occurs from the start, then the connections to the power supply are reversed |
Change the connections to the power supply |
If this occurs after some time into a long run, then the tank buffer is depleted |
If tank buffer is exposed to electrical current for a long time and the tank buffer is not recirculated, then the pH of the buffer changes near the electrodes (because protons are being reduced to hydrogen gas at one electrode, and hydroxide ions are being oxidized to water and oxygen at the other). When this happens, the dye will become positively charged relative to the more negative environment at the anode, and start migrating in the "wrong" direction (i.e., to the cathode). |
Poor band resolution
可能原因 | 建议的补救措施。 |
---|---|
Poor quality of reagents |
Use only the highest quality reagents |
Issues related to Urea |
Only use freshly deionized urea. |
Low molecular weight species have diffused |
Begin electrophoresis as soon as the sample is loaded to prevent low molecular weight species from diffusing. |
Poor stacking |
1. Only use gels that were recently prepared |
Incomplete gel polymerization |
Allow the gel to polymerize fully |
Running conditions |
Conduct the separation at a lower current or voltage setting |
Too much TEMED or APS (Ammonium persulphate) |
Lower the amount of TEMED or APS |
Sample incorrect prepared |
Sample preparation: a. Dialyze or desalt the sample. b. Centrifuge or filter sample before loading to remove particulates c. Reduce the sample volume or concentration. d. Improve dissociation of protein subunits by heating sample in SDS (Sodium dodecyl sulfate) sample buffer 1-2 minutes at 100 °C. Store on ice after heating. e. Store sample on ice before it is denatured. f. Add protease inhibitors such as PMSF (Phenylmethyl-sulfonyl fluoride) if necessary to prevent proteolytic degradation of sample. g. Add more mercaptoethanol or dithiothreitol; check sample treatment. h. Make fresh SDS sample buffer i. Use the same buffer for the sample as for the stacking gel. j. Increase the amount of glycerol or sucrose to increase sample density. k.Store samples to be frozen in aliquots to prevent repeated freezing and thawing. l. In a continuous buffer system, the sample may be too dilute. Use a discontinuous buffer system with a stacking gel, or a more concentrated sample. |
Smiling at buffer front
可能原因 | 建议的补救措施。 |
---|---|
Too high running temperature |
Reduce the running temperature: |
Water is impure |
Use only double-distilled water |
Gels adhere to glass plate when opening sandwich
可能原因 | 建议的补救措施。 |
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Dirty plates e.g. fingerprints |
Soak plates in a strong laboratory detergent and rinse well in distilled water. Please use gloves. |
Plates are scratched |
Replace glass plates. To some extent the effect of scratches can be counteracted by treating plates with silanizing reagent such as Repel-Silane. |
Plates were stored with, or soaked together with, plates that were pre-treated with Bind-Silane. |
Always segregate Bind-Silanized plates. Bind-Silanized plates can "contaminate" untreated plates if placed in contact, or soaked together. Glass plates can be re-used after scraping off the polyacrylamide gel and thoroughly washing the glass plates with strong sodium hydroxide solution. |
Swirl patterns in gel
可能原因 | 建议的补救措施。 |
---|---|
Too much catalyst: gel polymerized in < 10 min |
Reduce both APS (Ammonium persulphate) and TEMED by 25%. |
Not enough catalyst: gel polymerized in > 50 min |
Increase both APS (Ammonium persulphate) and TEMED by 50% |
Solutions not mixed |
Mix thoroughly after adding TEMED |
Brittle gel
可能原因 | 建议的补救措施。 |
---|---|
Too much bisacrylamide. |
Crosslinker should be at 2.6 %C for standard SDS (Sodium dodecyl sulfate) gels where: %C = (g bis x 100)/(g monomer + g bis) |
Stained sample appears at or near the buffer front
可能原因 | 建议的补救措施。 |
---|---|
Gel concentration |
Near the buffer front. Molecules are not sufficiently restricted by the resolving gel pore size: increase the %T (Total acrylamide concentration). |
Protein has degraded |
Use protease inhibitors during the isolation step to prevent protein degradation by proteases. |
Protein has precipitated |
The protein has precipitated. Heat the sample at a lower temperature (70 °C or less) for 1–2 min. |
Protein bands are diffuse or broader than usual
可能原因 | 建议的补救措施。 |
---|---|
Incomplete polymerization |
Ensure that polymerization is complete |
pH of buffers is off which is why stacking is not occurring. |
Prepare fresh SDS (Sodium dodecyl sulfate) running buffer and gel buffers |
Sample has degraded |
To avoid or retard sample degradation, store on ice and fully denature the sample |
- |
Try the following: |
Stacking is not occurring due to incorrect buffer pH |
Prepare fresh SDS (Sodium dodecyl sulfate) running buffer and gel buffers |
Sample doesn't contain same buffer as stacking gel. |
Use the same buffer for the sample as for the stacking gel. |
Too much TEMED or APS (Ammonium persulphate) |
Reduce TEMED or APS concentration by 25% |
Discontinuity between both gels |
Prepare the separating gel surface by rinsing it with stacking gel monomer before pouring the stacking gel to ensure continuity between both gels |
Incomplete gel polymerization.
可能原因 | 建议的补救措施。 |
---|---|
Poor chemicals |
1. Use only recent stock of the highest quality reagents. |
Solutions with extreme pH values (especially acidic) may not polymerize. |
- |
Oxygen is present |
Remove oxygen from the gel environment: Degas the monomer solution 5 to 10 minutes before pouring and then overlay the gel surface with water-saturated n-butanol. |
Gel solution has too low temperature |
Adjust the gel solution temperature to a minimum of 20 °C, especially for low %T (Total acrylamide concentration) gels. |
Too low concentration of TEMED or APS (Ammonium persulphate) |
Increase TEMED or APS concentration, or both |
Incomplete transfer / Molecules "blow through" the membrane
可能原因 | 建议的补救措施。 |
---|---|
Field strength too high |
Decrease the field strength |
Transfer period too long |
Decrease the transfer period |
- |
Include methanol in the protein transfer buffer (up to 20% for nitrocellulose, or up to 10% for PVDF). PVDF=Polyvinylidene difluoride |
Overheating
可能原因 | 建议的补救措施。 |
---|---|
Water in the outside tank too warm or no water in tank |
1. Fill the outside tank with cold water at the start of the transfer? There is no active cooling with this unit, and thus temperature should always be monitored. |
Set current is too high |
Since the miniVE blot module's cross-sectional area is small (roughly 85 cm2) you would use less current to get the same amount of transfer as larger instruments e.g. TE42. 150 mA (or less) for 2 hr should be sufficient. For instance to blot at 400 mA for 2 hr using a miniVE would result in smaller and medium-sized proteins blowing through the membrane. |