![]() ![]() Go back to the original non-passaged cell line and run the current and original cell line samples in parallel. Problem: Multiple/Extra bands Cell lines that have been frequently passaged, gradually accumulate differences in their protein expression profiles If the target is too strong, wait for 5-10 minutes and re-expose to film. Make sure that the membrane does not dry out during incubation. PVDF is considered to give higher background than nitrocellulose membrane The choice of membrane may give a high background Increase the Tween-20 concentration in the wash-buffer (0.1%-0.5%). The washing of unbound antibodies may be insufficient Substitute the milk with 3% BSA as blocking reagent. Non-fat dry milk may contain the target antigen (phosho-specific protein) ![]() Run a secondary control without the primary antibody, choose an alternative secondary antibody if bands developĪdd a mild detergent such as Tween-20 to the incubation and the washing buffer. The secondary antibody may be binding nonspecifically or reacting with the blocking reagent Titrate the antibody to the optimal concentration, incubate for longer time but in more diluted antibody solution (a slow but targeted binding is best). Use mono-specific or antigen affinity-purified antibodies. The primary antibody may be binding non-specifically or the concentration of the primary antibody may be too high Adjust the concentration of the blocking reagent up or down as needed. Increase the blocking incubation period and consider changing the blocking reagent. Problem: High background The blocking of non-specific binding might be absent or insufficient Instead of using 5% milk in the antibody buffers try removing the milk or using 0.5%, or substitute with 3% BSA. If no color develops or if it is weak make fresh reagents.ĭecrease the milk percentage in blocking buffer and in the antibody solutions. Mix the enzyme and its substrate in a tube. 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. 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. 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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