| Causes | Solutions |
| Membrane fouling | Use clean tweezer and operate with gloves to prevent membrane fouling. |
| The membrane has dried out | Incubate in sufficient reaction solution to prevent the membrane from drying out. |
| Blocking insufficient | Increase the blocking incubation time |
| Inappropriate blocking buffer | Switch different blocking buffer |
| Buffer solution has been contaminated | Use new buffer solution |
| Incomplete washing | Increase washing time and washing buffer’s volume |
| The antibody concentration may be too high | Decrease the concentration of primary antibody or secondary antibody |
| Cross-reaction between antibody and blocking agent | Choose blocking solution without cross-reaction. Add Tween-20 to the washing buffer to reduce cross reaction. |
High background: |
Improved: |
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| Causes | solutions |
| Insufficient antigen | Increase amount of loading samples |
| Protein degradation | Re-prepare samples |
| The antigen is blocked by blocking buffer | Optimize blocking solution, decrease blocking time or decrease the concentration of proteins in the blocking solution. |
| No or low level of target protein in samples | Run a positive control. If the level of target protein in samples is low, try to increase amount of loading sample. |
| Wrong choice of membrane | Choose suitable pore size membrane. Use 0.45um size membrane for proteins larger than 22KD. Use 0.2 µm size membrane for proteins smaller than 22 KD. |
| Poor transfer of protein to membrane | Make sure there are no air bubbles between the gel and membrane during transfer. Always ensure assembling electrode correctly. Control transfer temperature and optimize transfer electricity and time. |
| Methanol concentration may be too high. | Too high concentration of methanol may result in the separation of protein and SDS and thus cause protein precipitation in the gel. At the same time, it may cause shrinking or hardening of the gel to inhibit transferring of high molecular weight proteins. As a result, choose suitable methanol concentration according to different molecular weight. |
| Excessive washing of membrane | Reduce washing time and washing times |
| Over blocking | Lower the concentration of your blocking solution and shorten blocking time. Change blocking solutions. |
| The primary antibody is inactive | Use effective antibody in expiration, avoid freezing- thawing repeatedly, and use fresh solution. |
| Insufficient reaction of antibody to membrane | Increase the concentration of the antibody and the incubation time. |
| The reagents are not compatible with each other | Primary antibody and species, primary antibody and secondary antibody, or enzyme and substrate are not compatible. Setting loading control can validate the secondary detecting system. |
| HRP Inhibited | Avoid sodium azide in all solutions and containers |
| Enzyme or substrate is inactive. | Directly mix enzyme and substrate. If no color, the enzyme doesn’t work. Choose active conjugated reagent. Use fresh substrate. |
| Exposure time is too short | Increase the exposure time |
| Low or no signal | Improved |
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| Causes | Solutions |
| Amount of loading samples is too large | Decrease amount of loading samples |
| Protein degradation | Use fresh samples and use protein inhibitor |
| Cells were cultured too many passages to result in protein variation | Use primary cells or less passaging cells to run a control |
| Antibody Concentration is too high | Decrease the concentration of the primary or secondary antibody |
| Cross-reaction | Choose monoclonal antibody or affinity purified antibody to ensure antibody specificity |
| Existence of the different protein isoforms | Some proteins derived from the same gene have different isoforms. The size of every isoform protein is different. |
| Non-specific signal caused by the secondary antibody | Run a secondary antibody control or choose other secondary antibody |
| Substrate is too sensitive | Use suitable substrate |
| Exposure time is too long | Reduce the exposure time |
| Non-specific bands: | Improved: |
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| Causes | Solutions |
| Existence of dimer or polymer | Increase a process or intensity of protein denaturation |
| Modification of proteins | Modification of proteins, such as glycosylation or phosphorylation, can result in an increase of molecular weight of protein. |
| Causes | Solutions |
| Air bubbles were trapped in the gap of gel and membrane during transferring. | Remove bubbles in the gap of gel and membrane when preparing for transferring. |
| Invisible dots on the bands: | Improved: |
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| Causes | Solutions |
| Substrate is not well-distributed during incubation | Even the substrate during incubation |
| Incomplete bands: | Improved: |
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| Causes | Solutions |
| Migration was too fast during electrophoresis | Reduce the voltage to slow down the migration |
| Migration was too hot | Run the gel in the cold room or in ice |
| Smile effect of the bands: | Improved: |
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| Causes | Solutions |
| HRP concentration is too high | Decrease the secondary antibody concentration |
| White bands on a black blot: | Improved: |
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| Causes | Solutions |
| Air bubbles were trapped against the membrane during transferring or the antibody is not well distributed during incubation | Try to remove bubbles. Keep shaking when incubating the antibody. |
| The blocking agent was not well dissolved. | Filter the blocking agent. |
| The antibody reacts with the blocking solution. | Choose suitable blocking solution |
| There are aggregates in the HRP conjugated secondary antibody. | Filter the secondary antibody agent and remove the aggregates. |
| Black dots on the blot: | Improved: |
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| Causes | Solutions |
| The antibody reacts with the MW marker | Add a blank lane between the marker and the adjacent sample lane |
