Possible Causes of Western Blotting and Recommendations for No Bands Verify that after successful transfer of the separated proteins to the membrane, slight Ponceau staining is observed. Reduce the dilution factor one to one or secondary antibody; Complete most reusable antibodies and try refreshing.
Western dive guide
Recommended download controls
Freeze SDS page
Transfer of proteins from gel to membrane
Immunoblot and detection
Cleaning and reprobing the membrane
What causes faint bands in SDS-PAGE?
Wavy, weak or sometimes diffuse bands may appear when protein is deficient, the positive binding of proteins to the membrane is low, there has always been pressure fluctuation between our own gel and the membrane during transfer or transfer, the protein a certain molecular weight is definitely optimized.
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The troubleshooting guide below is generally intended to explain the causes and possible solutions to common problems that occur when translating to the West.
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No signal or weak signal
Primary antibody concentration too low
Increase the actual concentration of primary antibodies (dtitration should help). The Novus Concentration Antibody Kit can be used to increase the concentration of primary antibodies.
Raise the hatching specimen to 4°C overnight.
If the parent antibody is reused too many times, any effective antibody concentration may be too low; use fresh antibody to amplify the signal
Target protein concentration is also low
Loading more protein into the well (titration can probably help)
Use a positive activity lysate that expresses the target protein, an overexpressing lysate, or a recombinant protein.
Ensure that the lysis buffer is one of the optimal buffers for the target protein; The lysis buffer differs depending on the center of protein localization.
If necessary, use immunoprecipitation or fractionation (such as atomic fractionation) to increase the concentration of a protein that is not abundant.
Include protease inhibitors in lysis buffer
Make sure that a particular samplenot spoiled.
Transfer of proteins from the gel to the membrane ultimately failed
If necessary, confirm that the proteins have been successfully transferred to the membrane by staining the membrane with Ponceau or Coomassie stains, pointing to the gel.
Confirm the same transfer by reading the download control printout.
Primary and legitimate antibodies are incompatible
Make sure that the additional anti-species antibody was generated at the same time as the primary one.
(for example, if the primary antibody was obtained from mice, in this case, use an anti-mouse secondary antibody)
The choice of membrane was not ideal
Check the hydrophobicity/hydrophilicity of the antigenic sequence. PVDF membranes may work better with hydrophilic/polar/charged antigens, although nitrocellulose may work better with hydrophobic/non-polar antigens
There are blocking issues
Blocking too long can mask many epitopes and inhibit antibody binding; Reduce lock incubation or focus on time resolvedblocking.
Go for a unique blocking solution
Excessive washing of the membrane
Detection reagents may become inactive over time. Make sure the reagents are fresh. An alternative antibody can be tested by applying it to the membrane and incubating the blot with the detection reagent.
Use much more sensitive reagents when working with proteins at low concentrations (titration may help; use ultrapure water to dilute).
Can you overload Western blot?
The vast majority of the IP antibody can non-specifically capture the recognition antibody. IP with the minimum amount normally associated with antibodies to avoid gel overload and simply use extended incubation if recommended. We recommend using no more than 10-20 µg of IP antibodies in approximately each lane/well to avoid overload.
Intro image was too short
Increase the number of days of exposure (test several times to determine optimal exposure time).
The antibody only recognizes native proteins
Really use reconstituted and denatured proteins if you make a living with antibodies that only detect native proteins.
Targets have a lower molecular weight than standard targets
Reduce transfer time to avoid excessive transfers. Wet blotting is recommended for secondaryth proteins, as well as for membranes with smaller pore sizes (0.2 μm vs. 0.45 μm)
Sodium azide causes bacteria
Sodium azide (often used to store generic antibodies) inhibits HRP activity. Make sure the laundry is washed enough to remove the sodium azide, or use azide-free sea salt pads.
High uniform background
Slightly increase blocking time and/or temperature.
Increase reagent concentration (try increasing reagent concentration to 10%).
Consider replacing blocking agent (milk instead of BSA)
Include optional blockers contained in antibody buffers (can also be increased as a percentage).
Not recommended for detection of phosphorylated proteins (milk and therefore casein are rich in phosphoproteins)
Non-specific binding to ensure high concentration of antibodies
Reduce the concentration with the main or secondary solution (may help)titration)
Contains an antibody buffer blocking agent.
Confirm the indication of the secondary antibody by manipulating the secondary antibody: omit the primary antibody and incubate the blot with the legitimate antibody only.
Insufficient washing of unbound antibodies
Increase the number and/or duration of washes.
Make sure the membrane never dehydrates during the Western Blot protocol.
Why are my western blots not working?
You may have used a completely differentClear filter settings for detection. Make sure your family has set up the device to measure the correct wavelengths. In fact, the exposure time may not be enough when shooting this particular location. Try rendering the spot with a longer exposure time.
Film adhesion too long
Shorten the exposure time (may be needed to test a wide range of exposure times).
Detection reagents are actually too sensitive
Dilute the detection reagent in clean water, or use a less sensitive detection reagent.
Stripes missing/wrong size or possibly multiple stripes
Target protein below non-specific binding threshold
Loading more and more proteins into SDS-PAGE of the actual gel
Enrich low-content immunosuppressive proteinsPrecipitation by fractionation
Use fresh lysates
Keep sample on ice just before sample buffer activator and boil.
Always include protease inhibitors in combination with phosphatase inhibitors when a phosphorylated target is detected.
Other health protein isoforms may be present
Alternative splicing, also known as multimer formation, can be useful to you. This may require an isoform-specific antibody.
Sometimes post-translational modifications may be present
The predicted molecular weight can be improved by many factors, such as the use of glycosylation, phosphorylation, and protein processing (assuming cleavage from preform to form). To confirm specificity, run positive controls, such as a recombinant lysate or sometimes protein overexpressing lysate, knockdown/knockout downexpressing lysate, in addition to negative controls .
Mottled or swirling background
Incorrect handling of the membrane
Minimize contact in combination with the membrane. Use clean tools to do something with the membrane.
Create new stamps
Flip the skin gels and the membrane between the ampoules completely before transferring.
Filter these secondary antibodies with a 0.2 filter to remove aggregates.