<http://schema.org/articleBody>
"For further assistance, please contact our technical service department.\r\n\r\n\r\n\tObservation: No Bands Observed\r\n\tObservation: Faint Bands (Weak Signal)\r\n\tObservation: Extra Bands\r\n\tObservation: High Background\r\n\tObservation: Diffuse Bands\r\n\tObservation: White Bands (ECL method)\r\n\tObservation: Patchy uneven spots all over the blot\r\n\r\n\r\n\r\n\r\n&amp;nbsp;\r\n\r\nObservation: No Bands Observed\r\n\r\n\r\n\r\n\t\r\n\t\t\r\n\t\t\tPossible Source\r\n\t\t\tSuggestion\r\n\t\t\r\n\t\t\r\n\t\t\tInsufficient antibody\r\n\t\t\tAntibody may have low affinity to protein of interest. Increase antibody concentration (2-4 fold higher than recommended starting concentration).\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tAntibody may have lost activity. Perform a Dot Blot.\r\n\t\t\r\n\t\t\r\n\t\t\tInsufficient protein\r\n\t\t\tIncrease the amount of total protein loaded on gel.\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tConfirm the presence of protein by another method.\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tUse a positive control (recombinant protein, cell line or treat cells to express analyte of interest).\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tPerform a Dot Blot.\r\n\t\t\r\n\t\t\r\n\t\t\tPoor transfer\r\n\t\t\tWet PVDF/Immobilon-P membrane in methanol or nitrocellulose membrane in transfer buffer.\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tEnsure that there is good contact between PVDF membrane and gel.\r\n\t\t\r\n\t\t\r\n\t\t\tIncomplete transfer\r\n\t\t\tOptimize transfer time. High MW protein may require more time for transfer.\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tTo ensure transfer is complete, stain the membrane with Ponceau S, Amido Black or India Ink.\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tUse prestained MW marker.\r\n\t\t\r\n\t\t\r\n\t\t\tOver transfer\r\n\t\t\tReduce voltage or time of transfer for low molecular weight proteins (&amp;lt; 10 kDa).\r\n\t\t\r\n\t\t\r\n\t\t\tIsoelectric point is &amp;gt;9\r\n\t\t\tUse alternative buffer system with higher pH such as CAPS (pH 10.5).\r\n\t\t\r\n\t\t\r\n\t\t\tIncorrect secondary antibody used\r\n\t\t\tConfirm host species and Ig type of primary antibody.\r\n\t\t\r\n\t\t\r\n\t\t\tOld antibody\r\n\t\t\tIf antibody is expired or past manufacturer warranty, purchase fresh antibody.\r\n\t\t\r\n\t\t\r\n\t\t\tIncorrect storage of antibodies\r\n\t\t\tFollow manufacturer's recommended storage and avoid freeze/thaw cycles.\r\n\t\t\r\n\t\t\r\n\t\t\tSodium Azide contamination\r\n\t\t\tMake sure buffers do not contain Sodium Azide as this can quench HRP signal.\r\n\t\t\r\n\t\t\r\n\t\t\tInsufficient incubation time with primary antibody\r\n\t\t\tExtend incubation time to overnight at 4\u00B0C.\r\n\t\t\r\n\t\r\n\r\n\r\n\r\n\r\n\r\n&amp;nbsp;\r\n\r\nObservation: Faint Bands (Weak Signal)\r\n\r\n\r\n\r\n\t\r\n\t\t\r\n\t\t\tPossible Source\r\n\t\t\tSuggestion\r\n\t\t\r\n\t\t\r\n\t\t\tLow protein-antibody binding\r\n\t\t\tReduce the number of washes to minimum.\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tReduce NaCl concentration in Blotting Buffer used for wash steps (recommended range 0.15M - 0.5M).\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tReduce NaCl concentration in Antibody Solution (recommended range 0.15M - 0.5M).\r\n\t\t\r\n\t\t\r\n\t\t\tInsufficient antibody\r\n\t\t\tAntibody may have low affinity to protein of interest. Increase antibody concentration (2-4 fold higher than recommended starting concentration).\r\n\t\t\r\n\t\t\r\n\t\t\tInsufficient protein\r\n\t\t\tIncrease the amount of total protein loaded on gel.\r\n\t\t\r\n\t\t\r\n\t\t\tInactive conjugate\r\n\t\t\tMix enzyme and substrate in a tube. If color does not develop or, it is weak. Make fresh or purchase new reagents. Switch to ECL.\r\n\t\t\r\n\t\t\r\n\t\t\tWeak/Old ECL\r\n\t\t\tPurchase new ECL reagents.\r\n\t\t\r\n\t\t\r\n\t\t\tNon-fat dry milk may mask some antigen\r\n\t\t\tDecrease milk percentage in Block and Antibody Solutions or substitute with 3% BSA.\r\n\t\t\r\n\t\r\n\r\n\r\n\r\n\r\n\r\n&amp;nbsp;\r\n\r\nObservation: Extra Bands\r\n\r\n\r\n\r\n\t\r\n\t\t\r\n\t\t\tPossible Source\r\n\t\t\tSuggestion\r\n\t\t\r\n\t\t\r\n\t\t\tNon-specific binding of primary antibody\r\n\t\t\tReduce primary antibody concentration. (See Figure 1)\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tReduce the amount of total protein loaded on gel.\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tUse monospecific or antigen affinity purified antibodies (such as R&amp;amp;D Systems \"MAB\" or \"AF\" designated antibodies).\r\n\t\t\r\n\t\t\r\n\t\t\tNon-specific binding of secondary antibody\r\n\t\t\tRun a control with the secondary antibody alone (omit primary antibody). If bands develop choose an alternative Secondary Antibody.\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tUse monospecific or antigen affinity purified antibodies (such as R&amp;amp;D Systems \"BAF\" or \"HAF\" designated secondary antibodies).\r\n\t\t\r\n\t\t\r\n\t\t\tNon-specific binding of primary or secondary antibodies\r\n\t\t\tAdd 0.1 - 0.5% Tween\u00AE 20 to primary or secondary Antibody Solution.\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tUse 2% non-fat dry milk in Blotting Buffer as a starting point to dilute primary and secondary antibodies. Adjust antibody concentration up or down as needed. (See Figure 2a &amp;amp; 2b)\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tIncrease number of washes.\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tIncrease NaCl concentration in Blotting Buffer used for antibody dilution and wash steps (recommended range 0.15M - 0.5M). (See Figure 2c)\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tIncrease Tween\u00AE 20 concentration in Blotting Buffer used for wash steps (0.1%-0.5%).\r\n\t\t\r\n\t\t\r\n\t\t\tAggregation of analyte\r\n\t\t\tIncrease amount of DTT to ensure complete reducing of disulfide bonds (20 -100mM DTT). Heat in boiling water bath 5-10 minutes before loading onto gel.\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tPerform a brief centrifugation.\r\n\t\t\r\n\t\t\r\n\t\t\tDegradation of Analyte\r\n\t\t\tMinimize freeze/thaw cycles of sample.\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tAdd protease inhibitors to sample before storage.\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tMake fresh samples.\r\n\t\t\r\n\t\t\r\n\t\t\tContamination of reagents\r\n\t\t\tCheck buffers for particulate or bacterial contamination. Make fresh reagents.\r\n\t\t\r\n\t\r\n\r\n\r\n\r\n\r\n\r\n&amp;nbsp;\r\n\r\nObservation: High Background\r\n\r\n\r\n\r\n\t\r\n\t\t\r\n\t\t\tPossible Source\r\n\t\t\tSuggestion\r\n\t\t\r\n\t\t\r\n\t\t\tNon-specific binding of primary antibody\r\n\t\t\tUse monospecific or antigen affinity-purified antibodies (such as R&amp;amp;D Systems \"MAB\" or \"AF\" designated antibodies).\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tBlock in 5% milk. Adjust the milk (2-5%) or NaCl (0.15-0.5M) concentrations of primary Antibody Solution. (See Figure 3).\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tDecrease antibody concentration.\r\n\t\t\r\n\t\t\r\n\t\t\tNon-specific binding of secondary antibody\r\n\t\t\tRun a control with the secondary antibody alone (omit primary antibody). If bands develop choose an alternative Secondary Antibody.\r\n\t\t\r\n\t\t\r\n\t\t\tInsufficient blocking\r\n\t\t\tStart with 5% dry milk with 0.1%- 0.5% Tween 20, 0.15 -0.5M NaCl in 25mM Tris (pH 7.4). Incubation time may be extended. Adjust milk concentration up or down as needed.\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tOvernight blocking at 4\u00B0C may decrease blocking efficiency since detergents might not be effective at lower temperatures.\r\n\t\t\r\n\t\t\r\n\t\t\tNon-fat dry milk may contain target antigen\r\n\t\t\tSubstitute with 3% BSA.\r\n\t\t\r\n\t\t\r\n\t\t\tNon-fat dry milk contains endogenous biotin and is incompatible with avidin/streptavidin\r\n\t\t\tSubstitute with 3% BSA.\r\n\t\t\r\n\t\t\r\n\t\t\tSome IgY antibodies may recognize milk protein\r\n\t\t\tSubstitute with 3% BSA.\r\n\t\t\r\n\t\t\r\n\t\t\tInsufficient wash\r\n\t\t\tIncrease number of washes.\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tIncrease Tween 20 concentration in Wash Buffer (0.1%-0.5%).\r\n\t\t\r\n\t\t\r\n\t\t\tNon-Specific Binding of Primary Antibody\r\n\t\t\tIncrease NaCl concentration in primary Antibody Solution and Blotting Buffer used for dilution of primary antibody and wash steps (recommended range 0.15M - 0.5M). (See Figure 4).\r\n\t\t\r\n\t\t\r\n\t\t\tFilm overexposed\r\n\t\t\tReduce exposure time.\r\n\t\t\r\n\t\t\r\n\t\t\t&amp;nbsp;\r\n\t\t\tIf target signal is too strong wait 5-10 minutes and re-expose to film.\r\n\t\t\r\n\t\r\n\r\n\r\n\r\n\r\n\r\n&amp;nbsp;\r\n\r\nObservation: Diffuse Bands\r\n\r\n\r\n\r\n\t\r\n\t\t\r\n\t\t\tPossible Source\r\n\t\t\tSuggestion\r\n\t\t\r\n\t\t\r\n\t\t\tExcessive protein on gel\r\n\t\t\tReduce amount of protein loaded\r\n\t\t\r\n\t\r\n\r\n\r\n\r\n\r\n\r\n&amp;nbsp;\r\n\r\nObservation: White Bands (ECL method)\r\n\r\n\r\n\r\n\t\r\n\t\t\r\n\t\t\tPossible Source\r\n\t\t\tSuggestion\r\n\t\t\r\n\t\t\r\n\t\t\tExcessive signal generated\r\n\t\t\tReduce antibody or protein concentration. Excessive antibody or protein can cause extremely high levels of localized signal (usually at a single band). This results in rapid, complete consumption of substrate at this point. Since there is no light production after the completion of this reaction, white bands are the result when exposed to film.\r\n\t\t\r\n\t\t\r\n\t\t\r\n\t\r\n\r\n\r\n\r\n\r\n\r\n&amp;nbsp;\r\n\r\nObservation: Patchy uneven spots all over the blot\r\n\r\n\r\n\r\n\t\r\n\t\t\r\n\t\t\tPossible Source\r\n\t\t\tSuggestion\r\n\t\t\r\n\t\t\r\n\t\t\tContamination of reagents\r\n\t\t\tCheck buffers for particulate or bacterial contaminate. Make fresh reagents.\r\n\t\t\r\n\t\t\r\n\t\t\tNot enough solution during incubation or washing\r\n\t\t\tMake sure membrane is fully immersed during washes and antibody incubations.\r\n\t\t\r\n\t\t\r\n\t\t\tAir bubble trapped in membrane\r\n\t\t\tGently remove any air bubbles. Especially during transfer.\r\n\t\t\r\n\t\t\r\n\t\t\tUneven agitation during incubations\r\n\t\t\tEnsure uniform agitation by placing on a rocker/shaker.\r\n\t\t\r\n\t\t\r\n\t\t\tContaminated equipment\r\n\t\t\tMake sure that the electrophoresis unit is properly washed. Protein or pieces of gel remaining on the unit may stick to the membrane. Wash membrane thoroughly.\r\n\t\t\r\n\t\t\r\n\t\t\tHRP aggregation\r\n\t\t\tFilter conjugate to remove HRP aggregates.\r\n\t\t\r\n\t\t\r\n\t\t\tLong exposure\r\n\t\t\tReduce exposure time.\r\n\t\t\r\n\t\r\n\r\n\r\n\r\nTween 20 is a registered trademark of ICI Americas.\r\n"^^<http://www.w3.org/2001/XMLSchema#string>
<https://www.rndsystems.com/resources/technical/troubleshooting-guide-western-blot>