小中大
一抗是否有postive control cell lysates如果有，和你的样品一起上样，做Western blot, 就能很快知道原因。其实WB no signal 的原因非常多，下面是我随便google一下在Abcam网站上查到的可能原因，希望对您有所帮助。good luck!
Possible reasons for No signal
1The primary antibody and the secondary antibody are not compatible.
Use secondary antibody that was raised against the species in which the primary was raised (e.g primary is raised in rabbit, use anti-rabbit secondary).
2Not enough primary or secondary antibody is bound to the protein of interest.
Use more concentrated antibody. Incubate longer (e.g. overnight) at 4ºC.
3Cross-reaction between blocking agent and primary or secondary antibody.
Use a mild detergent such as Tween20 or switch blocking reagent (i.e. commonly used blocking reagents are milk, BSA, serum or gelatin).
4 The primary antibody does not recognize the protein in the species being tested.
Check the datasheet or perform a ClustalW alignment to ensure your antibody should react with the target protein; Run the recommended positive control.
5Insufficient antigen.
Load at least 20-30 μg protein per lane; Use protease inhibitors; Run the recommended positive control.
6 The protein of interest is not abundantly present in the tissue.
Use an enrichment step to maximize the signal (e.g. prepare nuclear lysates for a nuclear protein, etc.).
7 Poor transfer of protein to membrane.
Check the transfer with a reversible stain such as Ponceau S; check that the transfer was not performed the wrong way; if using PVDF membrane make sure you pre-soak the membrane in MeOH then in transfer buffer.
8 Excessive washing of the membrane.
Do not over wash the membrane.
Too much blocking does not allow you to visualize your protein of interest.
9 Instead of using 5% milk in the antibody buffers try removing the milk or using 0.5%. Switch blocking
reagents or block for less time.
10 Over-use of the primary antibody.
Use fresh antibody as the effective concentration is lowered upon each re-use.
11 Secondary antibody inhibited by sodium azide.
Do not use sodium azide together with HRP-conjugated antibodies.
12 Detection kit is old and substrate is inactive.
Use fresh substrate.
