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Trouble shooting guide
Trouble shooting guide
| PRIOR TO ELECTROPHORESIS | |||
| Upper buffer chamber leaks | Upper buffer chamber over filled Improper assembly of the upper buffer chamber 6 | Drain the chamber, dry the top corners of the chamber and fill to a few mm below the maximum level. Check assembly of gel cassettes in apparatus. | |
| The pH of the running buffer deviates from the required value by 0.2 pH units | Buffer incorrect 2 | DON’T ADJUST THE pH - Check buffer protocol and dilution; prepare fresh buffer and try again. | |
| Sample floats out of sample well or fails to form a layer at the bottom of the well | Running buffer too concentrated or incorrect 2 Glycerol or sucrose was omitted or is insufficient in sample buffer | Check buffer protocol and dilution; prepare fresh buffer and try again. Insure of thorough mixing especially when diluting stock solutions. Add glycerol or sucrose to sample buffer. | |
| Sample buffer is yellow in colour | Solution acidic Too little bromophenol blue in sample buffer | Add NaOH until the solution turns blue. Make fresh sample buffer with BPB concentration of 0.005-0.01 %. | |
| Current is much higher than the expected start current | Transfer buffer too concentrated | Prepare fresh buffer or dilution. Make sure Tris base was used and not Tris-HCl when transfer solution was made. | |
| Current is much lower than the expected start current | Buffer too dilute | Prepare fresh buffer or dilution. | |
| Power supply won’t allow to switch the unit “ON” / current reading on power supply is zero | Connection to power supply not complete Insufficient buffer level10 Ionic strength of the buffer is too high Power supply is operating at | Check connections between lid of apparatus and the electrode assembly jacks. Check connections between lid of apparatus and power supply. Make sure the upper buffer chamber doesn’t leak. Verify if assembly was properly done. Make sure the level of buffer in the upper and lower buffer chamber is sufficient. Check buffer protocol and dilution; prepare fresh buffer and try again. Use a power supply with higher limits. | |
| | a current close to its limit Broken electrode Blown fuse on power supply | | Check the apparatus to ensure the electrodes are intact. Check fuse and replace, if necessary. |
| Run too fast /poor resolution | Running buffer too dilute or incorrect 2 Power conditions excessive 1 | | Check running buffer recipe; prepare fresh buffer and try again. Insure of thorough mixing especially when diluting stock solutions. Decrease voltage or current by 25-50%. |
| Run time is too long | Power conditions insufficient Running buffer too concentrated or incorrect 2 Insufficient buffer level 10 | 1 | Increase voltage or current by 25-50%. Check buffer recipes and dilutions; remake buffer and try again. Insure of thorough mixing especially when diluting stock solutions. Make sure the upper buffer chamber doesn’t leak. Verify if assembly was properly done. Make sure the level of buffer in the upper and lower buffer chamber is sufficient. Upper buffer chamber overfilled Misassembly of the upper buffer chamber Desalt the sample and neighboring samples prior to resuspending in lower salt buffer or buffer with a different cation content. Use sample buffer of the same ionic strength as the gel. |
| Upper buffer chamber leaks 6 High salt concentration in sample 3 | |||
| ELECTROPHORESIS RESULTS | |||
| Bromophenol blue doesn’t sharpen into a concentrated zone in the stacking gel | High salt concentration in sample 3 | | Desalt the sample and neighboring samples prior to resuspending in lower salt buffer or buffer with a different cation content. Use sample buffer of the same ionic strength as the gel. |
| Diffuse tracking dye | Buffer stock solutions are too old Bromophenol Blue (BPB) concentration is too high | | Prepare fresh sample buffer. Maximum shelf life of the solution is 30 days at 4°C. Prepare fresh running buffer and dilution. Make fresh sample buffer with BPB concentration of 0.005-0.01 %. |
| Side lanes curl up / Gels “smiles” | Uneven / over heating of the gel 5 | | Turn down voltage or current setting by 25-50%. Check running buffer to ensure it was properly |
| | Leakage of buffer along sides Gel has expired | prepared, replace if necessary. Fill the tank / lower buffer chamber to its maximum level. Pre-chill the running buffer. Conduct electrophoresis in a cold room. Use direct cooling if your apparatus allows to recirculate coolant or cold tap water in the cooling core. Check the setup of the apparatus to ensure a secure seal with the gasket. Verify expiration date of gel. | |
| Poor resolution | Running buffer too dilute or incorrect 2 Sample volume too large and not sufficiently concentrated Power conditions excessive 1 Excess SDS 8 Gel expired Proteins not fully reduced 4 Protein degradation of sample 7 | Check buffer protocol and dilution; prepare fresh buffer and try again. Insure of thorough mixing especially when diluting stock solutions. Reduce the sample volume. Concentrate the sample. Decrease voltage or current by 25-50%. Do not exceed 200 microgram SDS per 30 microliter of sample. Verify expiration date of gel. Prepare fresh sample buffer solution using fresh ß-mercaptoethanol or dithiothrietol (DTT), fresh sample buffer should be prepared every 30 days. Heat sample in SDS sample buffer 1-2min. at 100C to improve dissociation of protein subunits. Increase ß-mercaptoethanol or DTT concentration in the sample buffer. Work at low temperatures; store sample on ice before it is denatured. Minimize the time between sample preparation and electrophoresis. When running denaturing gels, make sure the samples are being heated for 2min. at 100°C in the presence of agents such as SDS. Store on ice after heating. Store sample to be frozen in aliquots to prevent | |
| | | repeated freezing and thawing (store at –40C to – 80C). Use protease inhibitors like PMSF to prevent proteolytic degradation of sample. | |
| Diffuse protein bands | Power conditions insufficient 1 SDS or sample buffer too old Protein sample not equilibrated Problems in sample preparation | Increase voltage by 25-50%. Check running buffer to ensure it was properly prepared. Prepare fresh solutions. Equilibrate sample to running conditions. Make sure sample was heated in SDS sample buffer 1-2 min. at 100°C to improve dissociation of protein subunits. | |
| Bands widening near bottom of gel NOTE: gels | Depleting electrolytes in lower buffer Adjoining lanes are loaded with dissimilar samples | Increase volume of lower buffer. Try to load all sample wells with the same volume including any empty sample wells with sample buffer. | |
| Bands are narrower than sample wells / constriction of lanes at bottom of gel | High salt concentration in sample 3 | Desalt the sample and neighboring samples prior to resuspending in lower salt buffer or buffer with a different cation content. Use sample buffer of the same ionic strength as the gel. | |
| Lateral band spreading | Diffusion of sample out of the wells before the power was turned on Diffusion during migration through the stacking gel Low salt concentration in sample 3 | Minimize the time between sample application and power start-up. Increase voltage or current by 25% while sample is going through stacking gel. Use sample buffer of the same ionic strength as the gel. | |
| Skewed bands / distorted bands | Air bubbles in sample well High salt concentration in sample 3 Gel cassette is held in place too tightly 11 Uneven / over heating of the gel 5 | Before loading samples, displace the air bubbles by rinsing the wells with running buffer. Desalt the sample and neighboring samples prior to resuspending in lower salt buffer or buffer with a different cation content. Use sample buffer of the same ionic strength as the gel. Do not over-tighten the assembly of the gel with the electrophoresis device. Turn down voltage or current setting by 25-50%. Check running buffer to ensure it was properly prepared, replace if necessary. | |
| | Presence of precipitate / aggragate in sample prior to electrophoresis 9 The upper buffer chamber leaks 6 | Fill the tank / lower buffer chamber to its maximum level. Pre-chill the running buffer. Conduct electrophoresis in a cold room. Use direct cooling if your apparatus allows to recirculate coolant or cold tap water in the cooling core. Centrifuge or filter the sample to remove particulates before adding sample buffer. Dilute the sample. Prepare new sample buffer. Decrease the temperature at which the sample is prepared to 70°C or less and limit exposure to heat to 1-2 min. Centrifuge or filter the sample before loading to remove particulates. Upper buffer chamber overfilled Improper assembly of the upper buffer chamber | |
| Bands streaking | Sample overloaded / Sample too concentrated Proteins are running too fast Buffer stock solutions are too old High salt concentration in sample 3 Presence of precipitate / aggregate in sample prior to electrophoresis 9 | Dilute sample. Decrease sample volume loaded. Reduce voltage by about 25%. Prepare fresh sample buffer. Maximum shelf life of the solution is 30 days at 4°C. Prepare fresh running buffer and dilution. Desalt the sample and neighboring samples prior to resuspending in lower salt buffer or buffer with a different cation content. Use sample buffer of the same ionic strength as the gel. Dilute the sample. Centrifuge or filter the sample to remove particulates before adding sample buffer. Dilute the sample. Prepare new sample buffer. Decrease the temperature at which the sample is | |
| | Gel pore size is too small Proteins insufficiently loaded with SDS 8 Proteins not fully reduced 4 Protein degradation of sample 7 Poorly soluble or weakly charged particles in sample | prepared to 70°C or less and limit exposure to heat to 1-2 min. Centrifuge or filter the sample before loading to remove particulates. Choose a lower concentration of acrylamide (decrease % in resolving gel). Increase SDS concentration in sample buffer. SDS and/or sample buffer too old, prepare fresh sample buffer. Prepare fresh sample buffer solution using fresh ß-mercaptoethanol or dithiothrietol (DTT), fresh sample buffer should be prepared every 30 days. Heat sample in SDS sample buffer 1-2 min. at 100°C to improve dissociation of protein subunits. Increase ß-mercaptoethanol or DTT concentration in the sample buffer. Use tributylphosphine instead of DTT and iodoacetamide. Work at low temperatures; store sample on ice before it is denatured. Minimize the time between sample preparation and electrophoresis. When running denaturing gels, make sure the samples are being heated for 2 min. at 100°C in the presence of agents such as SDS. Store on ice after heating. Store sample to be frozen in aliquots to prevent repeated freezing and thawing (store at –40°C to –80°C). Use protease inhibitors like PMSF to prevent proteolytic degradation of sample. Change pH of buffers Check for carbohydrates, enzymatically remove, if necessary. | |
| Smear on the gel in each lane | Sample overloaded / Sample too concentrated Contaminating molecules (DNA, RNA, lipids, organic solvents, etc.) | Dilute sample. Decrease sample volume loaded. Run a lane with just the usual amount of sample buffer, to exclude it as the source of smearing. | |
| | | Try to use decreasing amounts of sample. Precipitate the sample to remove significant quantitites of nucleic acids, lipids, and salts. | |
| Two bands (doublet) where a single band is expected | Proteins not fully reduced 4 Uneven / over heating of the gel 5 | Prepare fresh sample buffer solution using fresh ß-mercaptoethanol or dithiothrietol (DTT), fresh sample buffer should be prepared every 30 days. Heat sample in SDS sample buffer 1-2 min. at 100°C to improve dissociation of protein subunits. Increase ß-mercaptoethanol or DTT concentration in the sample buffer. Use tributylphosphine instead of DTT and iodoacetamide. Turn down voltage or current setting by 25-50%. Check running buffer to ensure it was properly prepared, replace if necessary. Fill the tank / lower buffer chamber to its maximum level. Pre-chill the running buffer. Conduct electrophoresis in a cold room. Use direct cooling if your apparatus allows to recirculate coolant or cold tap water in the cooling core. | |
| Same protein bands across all gel tracks or observed in several neighboring lanes | Contamination of the sample buffer Sample overflow from one well has contaminated adjacent wells Gel cassette is held in place too tightly11 | Make new sample buffer. Use a Reduce the sample volume. Do not delay while loading wells A full well left next to an empty well would eventually contaminate the empty well over time. Do not over-tighten the assembly of the gel with the electrophoresis device. | |
| Artifact band observed at approx. 67kDa in reduced samples, especially after silver staining | Excess reducing agent (ß- mercaptoethanol) Skin protein contaminants | The addition of iodoacetamide to the sample buffer just before applying the sample to the gel has been shown to eliminate these artifact bands. Use new electrophoretic solutions and wear gloves when handling and loading the gel. More common when highly sensitive stains are used. | |
| Protein bands collect near the bottom of the gel | Gel pore size is too large Protein degradation of | Choose a higher concentration of acrylamide (increase % of resolving gel). Work at low temperatures; store sample on ice | |
| | sample 7 | before it is denatured. Minimize the time between sample preparation and electrophoresis. When running denaturing gels, make sure the samples are being heated for 2 min. at 100°C in the presence of agents such as SDS. Store on ice after heating. Store sample to be frozen in aliquots to prevent repeated freezing and thawing (store at –40°C to –80°C). | |
| | | Use protease inhibitors like PMSF to prevent proteolytic degradation of sample. | |
| Protein bands collect near the top of the gel when the dye front has reached the bottom of the resolving gel | Gel pore size is too small Presence of precipitate / aggregate in sample prior to electrophoresis 9 | Choose a lower concentration of acrylamide (decrease % of resolving gel). Decrease the temperature at which the sample is prepared to 70° C or less and limit exposure to heat to 1-2 min. Treat sample at lower temperature (60°C). Prepare new sample buffer. Work at low temperatures; store sample on ice before it is denatured. Minimize the time between sample preparation and electrophoresis. When running denaturing gels, make sure the samples are being heated for 2 min. at 100°C in the presence of agents such as SDS. Store on ice after heating. Store sample to be frozen in aliquots to prevent repeated freezing and thawing (store at –40°C to –80°C). Use protease inhibitors like PMSF to prevent proteolytic degradation of sample. | |
| Bands close in size do not separate well, or there is an overlap | Gel pore size is too large Sample volume too large and not sufficiently concentrated | Choose a higher concentration of acrylamide (increase % of resolving gel). Use shallow gradient gel or single concentration gel. Reduce sample volume Concentrate the sample | |
| Protein bands are not sufficiently resolved | Insufficient electrophoresis Gel pore size is inappropriate | Prolong the run. Choose a different concentration of acrylamide for the resolving gel. | |
| Insoluble material in sample, using denaturing (SDS-PAGE) conditions | Proteins insufficiently loaded with SDS 8 Proteins not fully reduced 4 Some samples aggregate on boiling pH of buffer is too low | Increase SDS concentration in sample buffer. SDS and/or sample buffer too old. Prepare fresh sample buffer solution using fresh ß-mercaptoethanol or dithiothrietol (DTT), fresh sample buffer should be prepared every 30 days. Heat sample in SDS sample buffer 1-2 min. at 100°C to improve dissociation of protein subunits. Increase ß-mercaptoethanol or DTT concentration in the sample buffer. Use tributylphosphine instead of DTT and iodoacetamide. Treat sample at lower temperature (60°C). Verify pH of solutions. If pH of running buffer wrong, prepare fresh solution (never adjust pH of running buffer). For other stock solutions, re‑ adjust pH. | |
| None or only a few proteins visible on the gel | Inappropriate sample extraction procedure (low protein concentration) | Perform protein assay (or SDS-PAGE) to estimate the protein concentration of the sample. | |
| More bands than expected observed for a purified protein | Protein degradation of sample 7 | Work at low temperatures; store sample on ice before it is denatured. Minimize the time between sample preparation and electrophoresis. When running denaturing gels, make sure the samples are being heated for 2 min. at 100°C in the presence of agents such as SDS. Store on ice after heating. Store sample to be frozen in aliquots to prevent repeated freezing and thawing (store at –40°C to –80°C). Use protease inhibitors like PMSF to prevent proteolytic degradation of sample. | |
| Irreproducibility of the protein bands pattern | Protein degradation of sample 7 | Work at low temperatures; store sample on ice before it is denatured. Minimize the time between sample preparation and electrophoresis. When running denaturing gels, make sure the samples are being heated for 2 min. at 100°C in the presence of agents such as SDS. Store on ice after heating. Store sample to be frozen in aliquots to prevent repeated freezing and thawing (store at –40°C to | |
| | | –80°C). | |
| | | Use protease inhibitors like PMSF to prevent proteolytic degradation of sample. | |
| High background of protein staining with indistinct protein bands | Proteins not fully reduced 4 Extensive sample proteolysis in native gels | Prepare fresh sample buffer solution using fresh ß-mercaptoethanol or dithiothrietol (DTT), fresh sample buffer should be prepared every 30 days. Heat sample in SDS sample buffer 1-2 min. at 100°C to improve dissociation of protein subunits. Increase ß-mercaptoethanol or DTT concentration in the sample buffer. Use tributylphosphine instead of DTT and iodoacetamide. Work at low temperatures; store sample on ice before it is denatured. Minimize the time between sample preparation and electrophoresis. Store sample to be frozen in aliquots to prevent repeated freezing and thawing (store at –40°C to –80°C). Use protease inhibitors like PMSF to prevent proteolytic degradation of sample. | |
| Diffuse background smear | Protein degradation of
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