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1.There is not enough primary or secondary antibody to bind to the target protein
Increase in the proportion of antibody dilution, or extend the incubation time of 4 degrees
2.The amount of antigen is insufficient or the protein sample is degraded
Increase in the amount of sample, or try to use fresh sample organization. And the addition of a protease inhibitor
3.Transfers are not sufficient
Use reversible colorants such as Pichiahong S to detect the effect of film transfer, check the transfer film operation is correct. PVDF membranes should be pre-immersed in methanol and then immersed in transfer buffer.
4.Excessive closure makes the target protein not colorable
Instead of 5% skimmed milk powder, use a 0.5% skim milk powder or an antibody dilution with no skimmed milk powder, or replace the sealant to reduce the closing time.
5.The primary antibody does not recognize the protein of the detection species
Reference to the instructions, compared to the immunogenic sequence and protein sequence to ensure that the antibody and the target protein will react, set a positive control.
6.The content of the target protein in the tissue is low
Concentrate to maximize the signal (for example, please use nuclear fission to detect nucleoprotein).
7.Over-washed the membrane
Do not over-wash the membrane
8.The secondary antibody is inhibited by sodium azide
Avoid the use of sodium azide and HRP labeled antibody. If the primary antibody contains sodium azide, please fully wash the film.
9.Detection kit expired and substrate deactivated
Use fresh substrate.
- No non-specific blocking or inadequate blocking: prolong the blocking time, consider replacing the appropriate sealant
- High primary antibody concentration: dilute the antibody to the appropriate concentration, incubation with higher dilution antibody longer (time-consuming but specific combination of the best).
- Antibody incubation temperature is too high: 4 ° C incubation.
- Primary or secondary antibodies have a cross-reactivity with the blocking agent: add mild detergents such as Tween-20 to the incubation and washing solution. Skim milk powder contains casein, the protein itself is a phosphorylated protein, will be combined with phosphorylated specific antibodies and easy to produce high background. Use BSA instead of milk powder as a blocking agent
- Unbound protein is not sufficient to wash: increase the number of washing
- Membrane selection results in high background: NC film is lower than PVDF membrane background
- Membrane drying: in the incubation process to prevent the film dry, at any step to ensure that the film has a sufficient reaction solution, stir into the stir constantly or gently shake the membrane immersed in the solution to avoid the phenomenon of dry film.
- Protein samples expressed in vivo are with a variety of modified forms such as acetylation, methylation, alkylation, phosphorylation, glycosylation, etc.: Read literature, use reagents to dephosphorylate samples, de-glycosylation to verify the post-translational modification.
- Protein sample degradation (decrease in protein molecular weight): Add enough protease inhibitor to the sample buffer
- Detection of uninformed new proteins or proteins with similar epitopes in the same family: Read other literature reports, or BLAST search, use recommended cell lines or tissues.
- High concentration of primary antibody often result in several protein bands: reduce antibody concentration and / or incubation time.
- High concentration of secondary antibody produce non-specific binding: reduce the antibody concentration, increase the secondary antibody control (without a primary antibody)
- Stripes are nonspecific bands: use blocking peptides to distinguish between specific and non-specific bands, only the specific band can be blocked to disappear.
- Target protein forms polymer: before loading to SDS-PAGE electrophoresis, incubate for 10 minutes instead of 5 minutes for depolymerization.