![]() Reduce the NaCl concentration in the blotting buffer of antibody solution. The number of washes should be reduced to a minimum. Problem: Faint Bands The protein-antibody binding is low Make sure that the buffers do not contain sodium azide when working with HRPconjugated antibodies. The secondary antibody is inhibited by sodium azide Use fresh antibody as the effective concentration is lowered upon each re-use. Switch blocking reagents or block for less time. Instead of using 5% milk in the antibody buffers try removing the milk or using 0.5%. Purchase fresh antibody when the antibody is expired or past the manufacturing date.įollow the manufacturer’s storage recommendations and avoid freeze/thaw cycles.īecause of too much blocking the protein of interest cannot be visualized The detection kit is old and the substrate is inactive Make sure that correct washing steps are included. Optimize the transfer time, high weight protein may require more time for transfer. Soak a nitrocellulose membrane in transfer buffer. If using PVDF membrane make sure you pre-soak the membrane in methanol then in transfer buffer. Check whether the transfer was not performed the wrong way. The transfer of the protein to the membrane is poorĬheck the transfer to ensure it is complete with Ponceau S, Amido Black or India Ink. ![]() The primary antibody does not recognize the protein in the species being testedĬheck the manufacturers datasheet to make sure that the antibody should react with the target protein. Use a mild detergent such as Tween-20 or switch the blocking reagent. Make sure that the antibodies have not exceeded their date of expiration.Ī cross-reaction between the blocking agent and primary or secondary antibody has taken place ![]() Run the recommended positive control.Įnsure that all antibodies have been stored correctly according to the manufacturer’s instructions. Confirm the presence of protein by another method (e.g. Increase the amount of protein that is loaded on the gel. If incubation time is insufficient, increase incubate time (e.g. The antibody may have lost its activity, perform a Dot Blot. The antibody has low affinity with protein of interest. Insufficient primary or secondary antibody is bound to the protein of interest primary is raised in rabbit, use secondary antibody raised in anti-rabbit). The primary antibody and the secondary antibody are not compatibleĪn incorrect secondary antibody is used, it might be raised against the species in which the primary was raised (e.g. ![]()
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