FAQ
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%.
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.
Which combs provide microtiter spacing for my Mighty Small electrophoresis unit?
The nine well comb and the 18 well comb provide microtiter spacing.
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.
Why is my electrophoresis run taking longer/shorter than usual?
1. Decrease the salt concentration of samples.
2. Adjust the solutions:
a. Check recipes, gel concentrations, and solutions. (For instance, do not use Tris-HCI instead of Tris base.)
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 exceeded, do not back-titrate. Prepare fresh buffer.
e. Dispose of older acrylamide solutions and use only stock of the highest quality.
f. Only use freshly deionized urea.
3. Adjust the voltage or current settings: To increase or decrease the migration rate, adjust the voltage or current by 25 - 50%.
4. Electrical leakage.
a. Check the alignment of the glass plates and spacers. Make sure there is no dirt or grease on the plates or spacers, and no cracks in the glass.
b. Check the heat-exchanger for leakage around the grommets.
5. Sample protein bands run slow but the marker bands are running fine.
a. The sample probably contains some nonionic (or cationic) detergent that is competing with the SDS (Sodium dodecyl sulfate) to coat the proteins. It is the negative charge of SDS that causes the proteins to migrate to the anode. Samples with Triton X-100 will lower the charge density and cause the sample proteins to migrate slower. The markers, coming out of a separate vial, probably don't have the Triton and will therefore migrate correctly. The solution is to clean the sample to remove the nonionic detergent (try TCA (Trichloroacetic acid)-acetone precipitation or the SDS-PAGE Clean-Up Kit).
Accessories
# | Product Name | Product Code | Price | |
---|---|---|---|---|
3 | Mighty Small Multiple Gel Caster, for SE 250 | 80614251 | 1,137.00 USD |
Add to cart
|
The instrument has a 4 mm recessed jack. Please see selection guide to see if you will require an adaptor.
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | EPS 301 Power Supply | 18113001 | 1,015.00 USD |
Add to cart
|
3 | Mains cable, 120 V | 19244701 | 71.49 USD |
Add to cart
|
4 | Mains cable 220 V | 19244801 | 110.80 USD |
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|
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | TE 22 Mini Tank Transfer Unit | 80620426 | 1,711.00 USD |
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# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | Glass plate, notched | 80613605 | 236.00 USD |
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2 | Glass plate, rectangular | 80613681 | 140.28 USD |
Add to cart
|
3 | Alumina plate, notched | 80613643 | 409.00 USD |
Add to cart
|
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | Spacer, t = 0.75 mm, L = 8 cm | 80613795 | 85.24 USD |
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2 | Spacer, t = 1.00 mm, L = 8 cm | 80613814 | 85.24 USD |
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|
3 | Spacer, t = 1.50 mm, L = 8 cm | 80613833 | 85.24 USD |
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|
Consumables
# | Product Name | Product Code | Price | |
---|---|---|---|---|
3 | PlusOne Glycine | 17132301 | 71.57 USD |
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4 | Tris | 17132101 | 120.41 USD |
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|
# | Product Name | Product Code | Price | |
---|---|---|---|---|
5 | Comb, no of wells = 10, t = 0.75 mm | 80613871 | 98.43 USD |
Add to cart
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6 | Comb, no of wells = 10, t =1.0 mm | 80613890 | 98.43 USD |
Add to cart
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7 | Comb, no of wells = 10, t =1.5 mm | 80613909 | 98.02 USD |
Add to cart
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8 | Comb, no of wells = 15, t = 0.75 mm | 80613947 | 98.43 USD |
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|
9 | Comb, no of wells = 15, t = 1.0 mm | 80613966 | 98.02 USD |
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10 | Comb, no of wells = 15, t =1.5 mm | 80613985 | 98.02 USD |
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# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | Sodium Dodecyl Sulfate | 17131301 | 75.15 USD |
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|
# | Product Name | Product Code | Price | |
---|---|---|---|---|
3 | Iodoacetamide | 25900066 | 165.10 USD |
Add to cart
|
5 | PlusOne Glycine | 17132301 | 71.57 USD |
Add to cart
|
# | Product Name | Product Code | Price | |
---|---|---|---|---|
2 | ECL DualVue™ Western Blotting Markers | 25190061 | 216.00 USD |
Add to cart
|
4 | Amersham ECL Full-Range Rainbow Molecular Weight Markers | 67610165 | 187.59 USD |
Add to cart
|
5 | Amersham ECL High-Range Rainbow Molecular Weight Markers | 67610166 | 154.85 USD |
Add to cart
|
6 | Amersham ECL Low-Range Rainbow Molecular Weight Markers | 67610167 | 154.85 USD |
Add to cart
|
7 | HMW Native Marker Kit | 17044501 | 345.00 USD |
Add to cart
|
9 | LMW-SDS Marker Kit | 17044601 | 345.00 USD |
Add to cart
|
Spare parts
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | Glass plate, rectangular | 80613681 | 140.28 USD |
Add to cart
|
2 | Alumina plate, notched | 80613643 | 409.00 USD |
Add to cart
|
3 | Adaptor kit (4 mm male / 4 mm female) | 80610527 | 50.52 USD |
Add to cart
|
4 | Comb, no of wells = 10, t = 0.75 mm | 80613871 | 98.43 USD |
Add to cart
|
6 | Gel seal | 80642143 | 38.86 USD |
Add to cart
|
7 | Spacer, t = 0.75 mm, L = 8 cm | 80613795 | 85.24 USD |
Add to cart
|
# | Product Name | Product Code | Price | |
---|---|---|---|---|
1 | Gasket | 80613719 | 64.42 USD |
Add to cart
|
2 | Gel seal | 80642143 | 38.86 USD |
Add to cart
|
# | Product Name | Product Code | Price | |
---|---|---|---|---|
4 | Gasket | 80613719 | 64.42 USD |
Add to cart
|
5 | Clamp with spring | 80614783 | 128.16 USD |
Add to cart
|
Troubleshooting
Find solutions to product related issues. For unlisted issues please contact local Cytiva service representation.
Bromophenol blue doesn't sharpen into a concentrated zone in the stacking gel.
Possible cause | Suggested remedy |
---|---|
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
Possible cause | Suggested remedy |
---|---|
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 |
Protein streaks vertically
Possible cause | Suggested remedy |
---|---|
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
Possible cause | Suggested remedy |
---|---|
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 |
Select symptoms
Possible cause | Suggested remedy |
---|---|
1. Avoid stressing the ears by only handling the sandwiches by the sides and bottom. |
Possible cause | Suggested remedy |
---|---|
Glass plates have been exposed to solutions with too high alkali concentration |
1. Avoid extended exposure of glass to alkali (high pH) solutions |
Possible cause | Suggested remedy |
---|---|
Clamp screws tightened too much, stressing the glass |
The screws need not be tightened more than finger-tight to ensure a seal |
Plates are stressed by excessive heat generated from the electrophoresis |
Run gels slower |
Foreign material lies on the spacers |
Check that no foreign material lies on the spacers before applying the clamps |
Possible cause | Suggested remedy |
---|---|
UBC is warped or shrunken which would happen if the unit had been run at temperatures exceeding 50 °C without proper cooling, or if the unit had been autoclaved |
Cooling requires an external cooling water recirculator, such as the MultiTemp III. |
Foam gasket doesn't seal correctly |
1. Check that the diameter of the foam gasket is 4.5 mm. Replace, if necessary |
Possible cause | Suggested remedy |
---|---|
The instrument has been exposed to anti-freeze or organic solvent |
Circulate only water or 50/50 water/ethylene glycol through the heat exchanger |
Unregulated pressure of coolant source |
Only use coolant sources with regulated constant pressure |
Too high buffer temperature, temperature above 45ºC |
All plastic parts are designed for a maximum temperature of 45ºC |
Possible cause | Suggested remedy |
---|---|
1. Check power supply. Try another power supply to verify that the problem does not lie with the power supply. |
Possible cause | Suggested remedy |
---|---|
Gels are not identical concerning % acrylamide, % crosslinking, buffer content or thickness |
Current splits evenly between the 2 gels only if the conductivity of the 2 gels are perfectly identical. |
Running buffers in the two upper chambers are not identical |
The running buffers in the two upper chambers must be identical |
Samples contribute differently |
Samples can contribute conductivity. High salt levels (or buffer concentrations) can increase the conductivity of one gel relative to the other. |
Possible cause | Suggested remedy |
---|---|
Solutions need to be adjusted |
Adjust the solutions: |
Too high salt concentration in samples |
Decrease the salt concentration of samples |
Particles in UBC |
Check the UBC. It should be free of particles. |
Electrical leakage |
1. Check the alignment of the glass plates and spacers. Make sure there is no dirt or grease on the plates or spacers, and no cracks in the glass. |
- |
Adjust the voltage or current settings: To increase or decrease the migration rate, adjust the voltage or current by 25 - 50%. |
Possible cause | Suggested remedy |
---|---|
Sample probably contains some nonionic (or cationic) detergent that is competing with the SDS (Sodium dodecyl sulfate) to coat the proteins |
It is the negative charge of SDS that causes the proteins to migrate to the anode. Samples with Triton X-100 will lower the charge density and cause the sample proteins to migrate slower. The markers, coming out of a separate vial, probably don't contain Triton and will therefore migrate correctly. The solution is to clean the sample to remove the nonionic detergent (try TCA-acetone precipitation or the SDS-PAGE Clean-Up Kit). TCA=Trichloroacetic acid |
Possible cause | Suggested remedy |
---|---|
Incorrectly prepared buffer, either in the buffer chambers or in the gel |
Make up fresh buffers and re-check gel casting recipe. |
Electrical leakage |
Check the alignment of the glass plates. Make sure there is no dirt or grease on the plates or spacers, and no cracks in the glass. |
Brittle gel
Possible cause | Suggested remedy |
---|---|
Too much bisacrylamide. |
Crosslinker should be at 2.6 %C for standard SDS (Sodium dodecyl sulfate) gels where: |
Poor band resolution
Possible cause | Suggested remedy |
---|---|
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
Possible cause | Suggested remedy |
---|---|
Too high running temperature |
Reduce the running temperature: |
Water is impure |
Use only double-distilled water |
Gels adhere to glass plate when opening sandwich
Possible cause | Suggested remedy |
---|---|
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
Possible cause | Suggested remedy |
---|---|
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 |
Stained sample appears at or near the buffer front
Possible cause | Suggested remedy |
---|---|
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. |
Incomplete gel polymerization.
Possible cause | Suggested remedy |
---|---|
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 |