The common problems that may occur in the fully automated protein immunoblotting processing system during experiments mainly include weak or no signal, high background, multi band phenomenon, abnormal bands, etc. The following is a detailed analysis of these problems:
Weak or no signal
Mismatch between primary and secondary antibodies: The secondary antibody needs to be of the same species as the primary antibody host, for example, if the primary antibody comes from a mouse, the secondary antibody should be an anti mouse antibody.
Insufficient antibody concentration: Use high concentration antibodies and extend the incubation time at 4 ℃, such as overnight incubation.
Cross reaction between blocking agents and antibodies: Use mild detergents such as Tween-20, or replace blocking agents (such as skim milk powder, BSA, serum, gelatin).
Proteins that do not recognize the test species in the primary antibody: Refer to the instructions, compare the immunogen sequence and protein sequence to ensure that the antibody and target protein will react, and set up a positive control.
Insufficient protein loading: The protein loading per lane generally does not exceed 20 μ g, and protease inhibitors are used with a positive control set up.
Insufficient membrane transfer: Use reversible staining agents to test the membrane transfer effect and check if the membrane transfer operation is correct. For example, PVDF membranes need to be pre immersed in methanol and then immersed in transfer buffer.
Primary antibody failure: Use fresh antibodies to avoid repeated use leading to a decrease in effective concentration.
Inhibition of secondary antibodies by sodium azide: Avoid using sodium azide and HRP labeled antibodies together.
Expired detection kit or substrate inactivation: Use fresh substrate.
High background
Lack of specific sealing or insufficient sealing: Extend the sealing time and consider replacing with a suitable sealing agent.
Excessive concentration of primary or secondary antibodies: Dilute the antibody to an appropriate concentration, or extend the incubation time with a higher dilution of the antibody.
Non specific binding or reaction between secondary antibody and blocking agent: Set up a secondary antibody control (without adding primary antibody).
Insufficient washing of unbound proteins: Increase the number of washes.
Membrane drying: During the incubation process, to prevent the membrane from drying out, it is necessary to ensure that the membrane has sufficient reaction solution at each step. The membrane can be immersed in the solution by continuously stirring or gently shaking it with a stirrer.
Improper membrane selection: Nitrocellulose membrane (NC membrane) has a lower background than PVDF membrane, and a suitable membrane can be selected according to experimental requirements.
Multi band phenomenon
Too many cell passages result in different protein expression: a parallel control experiment was conducted using the original non passaged cell line together with the current cell line.
The protein samples expressed in vivo have various forms of modification, such as acetylation, methylation, alkylation, phosphorylation, glycosylation, etc. Reagents can be used to dephosphorylate and deglycosylate the samples to demonstrate post-translational modifications.
Protein sample degradation: Add sufficient protease inhibitors to the sample buffer.
Detected new proteins that have not been reported before or proteins with similar epitopes but different structures within the same protein family: refer to other literature reports, or use BLAST to search for cell lines or tissues recommended in the instructions.
Excessive concentration of primary antibody: reduce antibody concentration and/or incubation time.
Strip abnormality
Uneven white spots on the background: there are bubbles or uneven distribution of antibodies on the membrane during membrane transfer. During the membrane transfer process, bubbles should be removed as much as possible, and shaking should be maintained during antibody incubation.
There are black spots on the background: the antibody binds to the blocking agent and can filter the blocking agent.
A white band appears in the deep background: if too much primary or secondary antibody is added, the concentration of the antibody should be diluted.
The staining of the target strip is too low/too high, and the separation is not complete: change the proportion of gel, use low concentration gel for protein with high molecular weight, and use high concentration gel for protein with low molecular weight.
Band "smile" effect: Migration too fast or electrophoresis temperature too high changes the pH value and migration speed, which can reduce migration speed or low-temperature electrophoresis (such as in cold storage or on ice).
Heterogeneous bands appear in the hybridization of the same protein: when preparing gel, the gel coagulates too fast, resulting in uneven proportion of acrylamide in the swimming lane. Refer to the formula of gel, add an appropriate amount of TEMED to the gel, and add an appropriate amount of 1% SDS (diluted with water) to the top of gel when placing to prevent the gel from drying.