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General 1D Electrophoresis

It is important to determine whether the problem is with the power supply, the apparatus, or the gel. Often, it helps to switch out the power supply or the lid to see if there is a faulty contact. Also, check to see whether the buffer core is damaged. Additionally, make sure there is sufficient buffer in the electrophoresis tank to cover the wells of the gel.

Here are possible causes and solutions:

Cause Solution
Buffers are too concentrated or incorrect Check buffer recipe; dilute or re-make if necessary.
Voltage, current, or wattage is set at a higher limit Decrease power conditions to recommended running conditions.

Here are possible causes and solutions:

Cause Solution
Sample overload Do not overload samples
Addition of reducing agent that is not fresh Reduce samples right before loading and do not use samples that have been stored in reducing agent
Re-oxidation of the protein during the run Add antioxidant to the running buffer if you are running NuPAGE™ gels
Presence of highly hydrophobic regions where the protein can exclude SDS Load the sample with 2X sample buffer instead of 1X
Excess salt in the sample Precipitate and reconstitute in lower salt buffer
Not enough SDS in the sample Add SDS to the upper buffer chamber (try 0.1%, 0.2%, 0.3% and 0.4%)

Barbell shaped bands are a result of loading too large of a sample volume. When a large sample volume is loaded, part of the sample tends to diffuse to the sides of the wells. When the run begins and the sample moves through the stacking portion of the gel, the sample will incompletely stack causing a slight retardation of the portion of the sample that diffused to the sides of the wells. This effect may be intensified for larger proteins, whose migration is more impeded in the low concentration acrylamide of the stacking gel. To alleviate the problem, we recommend concentrating the protein and loading a smaller volume. This gives a "thinner" starting zone.

"Smiling" bands may be the result of the acrylamide in the gel breaking down, leaving less of a matrix for the proteins to migrate. We recommend checking to ensure that the gels have not been used past their expiration date.

Gel lifting off the cassette can be caused by:

  • Expired gels that are degrading.
  • Improper storage of gels.
  • Too much heat accumulating during the electrophoresis run due to excessive current.
  • Insufficient polymerization of the polyacrylamide.

There may be too much β-mercaptoethanol, dithiothreitol (DTT) or sample buffer salts in your samples. If the proteins are over reduced, they can be negatively charged and actually repel each other across the lanes making the bands narrower as they progress down the gel.

A portion of the protein sample may have re-oxidized during the run, or may not have been fully reduced prior to the run. We recommend preparing fresh sample solution using fresh beta-mercaptoethanol or dithiothreitol (DTT). For NuPAGE™ gels, we recommend adding antioxidant to the running buffer.

Possible cause:

  • Poor polymerization around sample wells
  • High salt concentration in sample
  • Uneven gel interface
  • Excessive pressure applied to the gel plates when the gel is placed into the clamp assembly
  • Uneven heating of the gel
  • Insoluble material in the gel or inconsistent pore size throughout the gel
  • Air bubble during the run

Remedy:

  • Remove excess salt/other material by dialysis, Sephadex™ G-25 or any other desalting column or using an Amicon™ concentrator.
  • Either use a cooled apparatus or reduce the current at which electrophoresis is performed.

Possible cause:

  • Carry-over contamination of sample from one well into neighboring wells due to loading error 
  • Contaminated running buffer 
  • Gel casting error: malformed wells

Remedy:

  • Use a gel loading tip to load wells 
  • Reduce the sample volume 
  • Do not delay while loading wells 
  • Do not delay after the run, as proteins can diffuse horizontally; a full well left next to an empty well would eventually contaminate the empty well over time.

Possible cause:

  • Excess reducing agent (beta-mercaptoethanol) 
  • Skin protein contaminants (keratin)

Remedy:

  • The addition of iodoacetamide to the equilibration 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. This issue is more common when highly sensitive stains are used.

This could be due to a gel polymerization issue combined with incorrect sample preparation (final sample dilution less than 1X). Please try a different lot of the same gel and make sure that the sample is correctly prepared.

This could be due to:

  • Debris in the well
  • High salt in the sample (make sure that the slat concentration does not exceed 50–100 mM)
  • Running buffer issue
  • Gel casting error

Here are some suggestions:

  • Check the gel type/percentage of the gel that was used; depending on the gel type and/or percentage, all the bands may not be seen. For example, small molecular weight protein may not resolve on a very low-percentage gel whereas higher molecular weight protein bands may not resolve on a high-percentage gel.
  • Check whether the protein was stored properly - improper storage conditions will result in protein degradation.

Here are some suggestions:

  • Make sure that the correct amount of protein is loaded per lane; loading too much protein can cause smearing and this is a problem especially with silver stained gels.
  • Bands will not be as well resolved in low percentage gels; try using a higher percentage gel.
  • If the bands look smeary and non-distinct after a western transfer/detection, this may be due to the antibody being too concentrated. Follow the manufacturer’s recommended dilution or determine the optimal antibody concentration by dot-blotting.

 

  • While loading, take care to make sure that there is no cross-contamination from adjacent sample lanes.
  • Make sure that you don’t load too much protein; overloading can result in extra bands and this is a problem especially with silver stained gels.
  • Improper storage of the protein or repeated freeze/thawing can result in protein degradation.

Precipitation of the LDS or SDS at 4 degrees C is normal. Bring the buffer to room temperature and mix until the LDS/SDS goes into solution. If you do not want to wait for it to dissolve, you can store the sample buffer at room temperature.

Bolt™ Bis –Tris Plus Gels

Due to the current wedge-well design of our Bolt™ gels, excess polyacrylamide (likely to be present in the area of the comb) is a consequence of our efforts to ensure that all wells are completely formed. Most of this excess material can be removed by slowly pulling the comb straight up out of the gel wells, turning the gel upside down, and then gently flicking the excess gel into a trash receptacle. Efforts are underway to reduce this excess.

We recommend increasing the contrast between the sample well and the rest of the gel by marking the cassette at the bottom of the wells with a marker pen prior to placing the cassette in the electrophoresis tank.

Here are some likely reasons and remedies:

  • Leaking of buffer from the inside of the tank to the outside. Leaking is most likely due to inappropriate removal of cassettes, which can lead to the gasket not sitting properly and loss of a good seal. We recommend pushing the gasket down with the thumb.
  • Not covering the wells completely with the buffer. We recommend filling the tank up to the electrodes prior to inserting the gel cassettes. Complete filling of the tank not only prevent leaks from the inside to the outside but also acts as a heat sink.
  • Not removing the tape from one of the gel cassettes. Make sure that the tape is removed from both gel cassettes before inserting them into the tank.
  • Not placing the gel cassette all the way to the bottom of the tank prior to clamping it (this will create a bypass of the current at the bottom, so the gel will run slower or not run at all).

Here are possible causes and solutions:

Cause
Solution
Buffers are too dilute
Check buffer recipe; remake if necessary.
Buffer chamber is leaking
Make sure the cassette clamp is firmly seated, the gaskets are in place and the cassette clamp is locked.
Current is set too low
Set correct current.

Here are possible causes and solutions:

Cause Solution
Tape left on the bottom of the cassette
Remove tape from bottom of cassette.
Connection to power supply not complete
Check all connections with a voltmeter for conductance.
Insufficient buffer level
Make sure there is sufficient buffer in the electrophoresis tank to cover the wells of the gel.

Here are possible causes and solutions:

Cause Solution
Buffers are too concentrated or incorrect Check buffer recipe; dilute or remake if necessary.
Current is set at a higher limit

Decrease current to recommended running conditions.

It is likely that the Bolt™ gel cassette was inserted backwards into the unit (large plate facing the front and the wells facing the back) even though this is pretty difficult to do. When the gel is inserted backwards, the current flows from the bottom of the gel to the top, resulting in the samples running in the opposite direction. Reversing of the leads will switch the direction of the gel run, however, this will cause the current to flow from the anode to the cathode. The cathode electrode is made of stainless steel with platinum coating, and the anode electrode is made of platinum wire. Flow of electrons from the anode to the cathode will result in rusting of the steel core. On the other hand, when the leads are connected properly, the electrons flow from the cathode to the anode and the recipient of the electrons is the platinum wire that does not rust.

Note: When the gel cassette is inserted properly, the lettering (gel type, SKU and expiration date) printed on the cassette will be backwards (reads from right to left).

Here are some causes and solutions for wavy dye fronts:

Cause Solution
Difference in buffer level between the inner and outer buffer chambers Both buffer chambers must be filled up to the electrode with wells completely covered. This will not only prevent leaks from the inside to the outside but will also act a heat sink and prevent wavy dye fronts.
Using running buffer that was diluted more than 1X
We recommend using 1X running buffer.
Using old running buffer Make sure that the running buffer is fresh and don’t reuse the running buffer.

NuPAGE™ Bis-Tris and NuPAGE™ Tris-Acetate Gels

This can happen if a frozen NuPAGE™ gel was used. Please make sure that NuPAGE™ gels are stored at 4–25 degrees C and are not accidentally frozen. Check refrigerator settings and store the gels on lower shelves away from the freezer section and away from the condenser.

A forward smear indicates that the antibody was being reduced in the gel during migration. This could have been caused by the addition of antioxidant in the sample buffer or due to rearrangement of disulfide bonds during heating in NuPAGE™ Sample buffer. Make sure that the correct concentration of reducing agent is used in the sample buffer and do not add any antioxidant in the sample buffer.

We do not recommend using frozen NuPAGE™ gels as:

  • They may have become brittle and may crack either before or after the run, or even when in the staining solution.
  • May cause the formation of bubbles during the run and band distortion particularly in the center lanes.

RIPA buffer contains a lot of detergent and hence would not be compatible with NuPAGE™ gels.

For proteins larger than 100 kDa, we recommend pre-equilibrating the gel in 2X Transfer buffer (without methanol) containing 0.02–0.04% SDS for 10 minutes before assembling the sandwich and then transferring using 1X transfer buffer containing methanol and 0.01%SDS.

Here are possible causes and solutions:

Cause
Solution
Buffers are too dilute Check buffer recipe; remake if necessary.
Upper buffer chamber is leaking Make sure the buffer core is firmly seated, the gaskets are in place and the gel tension lever is locked.
Voltage is set too low Set correct voltage.

We recommend marking the cassette at the bottom of the wells with a marker pen prior to assembling the upper buffer chamber. Also, we recommend illuminating the bench area with a light source placed directly behind the XCell SureLock™ unit.

You may purchase the ZOOM™ adapters, Cat. No. ZA10001 to help you connect your leads to the power supply.

  • Double check that the tape on the bottom of the gel has been removed.
  • Make sure that the gel(s) are oriented so that the taller sides of the cassette (with the printing) are facing the outside of the electrophoresis unit.
  • Make sure that the inner buffer chamber is filled sufficiently so that the wells are covered with buffer. If the wells are not covered, check for leaks and reseal.
  • Double check to see if there are any loose electrodes or connections on the Mini cell unit.
  • Check the power supply unit.

Novex™ Tris-Glycine Gels

This can happen if Tris-Glycine gels are run using NuPAGE™ Running buffer containing Antioxidant. Please make sure that the correct Tris-Glycine Running buffer is used with Tris-Glycine gels.

Here are possible causes and solutions:

Cause
Solution
Buffers are too dilute Check buffer recipe; remake if necessary.
Upper buffer chamber is leaking Make sure the buffer core is firmly seated, the gaskets are in place and the gel tension lever is locked.
Voltage is set too low Set correct voltage.

We recommend marking the cassette at the bottom of the wells with a marker pen prior to assembling the Upper buffer chamber. Also, we recommend illuminating the bench area with a light source placed directly behind the XCell SureLock™ unit.

You may purchase the ZOOM™ adapters, Cat. No. ZA10001 to help you connect your leads to the power supply.

  • Double check that the tape on the bottom of the gel has been removed.
  • Make sure that the gel(s) are oriented so that the taller sides of the cassette (with the printing) are facing the outside of the electrophoresis unit.
  • Make sure that the inner buffer chamber is filled sufficiently so that the wells are covered with buffer. If the wells are not covered, check for leaks and reseal.
  • Double check to see if there are any loose electrodes or connections on the Mini cell unit.
  • Check the power supply unit.

It may be possible to reduce background by using the protocol provided for NuPAGE™ Novex™ Bis-Tris gels/Small Peptides. This protocol incorporates an extra fix step to remove excess SDS, which can act as an anti-colloidal agent and lead to higher background. The low pH of the staining solution will fix the gel, but not as fast as the pre-fix step specified in the NuPAGE™ protocol.

Background is generally higher in gels with less than 10% acrylamide percentage due to penetration and trapping of colloids within the large pores of these gels. Excess background may be reduced by incubating the gel in 25% methanol solution until a clear background is obtained. Be aware that the dye will also be partially removed from the bands and that prolonged incubation in >25% methanol will result in complete destaining of protein bands and background.

The most common cause of abnormally high current is the transfer buffer. If the transfer buffer is too concentrated, this leads to increased conductivity and current. High current may also occur if Tris-HCl is accidentally substituted for the Tris base required in the transfer buffer. This will again result in low buffer pH and lead to increased conductivity and current and subsequently, overheating. We recommend checking the transfer buffer and its reagent components and re-diluting or remaking the buffer.

Thermo Scientific™ Precise™ Protein Gels

Here are possible causes and solutions:

Cause
Solution
Air bubbles in the sample wells, or between gel and cassette, or at the bottom of the cassette.
Use a transfer pipette to displace the air bubbles from the sample wells.
Sample contains appreciable carbohydrate

Remove the carbohydrate by enzymatic or chemical means.
Sample contains lipoproteins Use a gel with large pore size at top. Try addition of a non-ionic detergent.

Here are possible causes and solutions:

Cause
Solution
Poorly soluble or weakly charged particles (such as carbohydrates) in sample Centrifuge samples.
Change pH of buffer.
Heat sample in the presence of SDS.

Here are possible causes and solutions:

Cause
Solution
Incorrect gel selection, sample overloading, and insufficient cooling buffer Select a gel that separates in the desired molecular weight range.
Reduce sample size.
Increase buffer volume in the outer tank.
For proteins of similar molecular weight a 2D separation may be required.

Here are possible causes and solutions:

Cause
Solution
Excess salt in the sample Reduce salt by dialysis or ultra-filtration.
Too much protein applied to the gel Optimize the amount of protein applied to the gel.

This could be due to excessive heating of the sample. We recommend using chilled buffer (<15 degrees C).

Here are possible causes and solutions:

Cause
Solution
SDS still present in the gel Wash the gels extensively (3 x 5 minutes) with ultrapure water and use 30% methanol to destain the gel.
Protein bands are diffusing Use 10% TCA to fix the proteins in the gel.

Please make sure that the Tris-HEPES SDS running buffer is used with these gels. The Precise™ Tris-HEPES gels are designed to be used with the Tris-HEPES SDS running buffer for optimal speed and resolution. We do not recommend using the Tris-Glycine SDS running buffer as this will result in poor migration and band resolution.