1. Spot 2 µl of samples onto the nitrocellulose membrane;
2. Let the membrane dry;
3.Block non-specific sites by soaking in 5% milk in TBS (0.5-1 hr, RT). Not adding tween-20;
4.Incubate with primary antibody dissolved in Milk/TBS for 30 min at RT;
5.Wash three times with TBS (3 x 30 min)[We just detect the protein with biotin label, so the secondary antibody is undesired ];
6.Incubate with ECL reagent for 1 min, cover with Saran-wrap (remove excessive solution from the surface), and expose X-ray film in the dark room. Try several different lengths of exposure.
What is wrong? Could you help me?I have dilute the protein sample to 20X, 40X, 60X, 80X 和100X.
I've never had this situation. If I didn't know anything and just watched at your film, I would guess you had an air bubble, but that's highly unlikely with 2ul spots...Sorry I'm not more helpful.
Hi, maybe this discussion can help you
https://www.researchgate.net/post/Can_anyone_help_me_with_my_dot_blot_analysis/1
When you detect by CL the white dot is usually related with too much protein. Although you diluted up to x100 the technique is very sensitive and can still be too big of protein amount
Hi, the white spots can either be due to oversaturation of the film by too much protein, as Fortunato pointed out, or by uneven loading of your protein solution. You could try higher dilutions, but since the white spot is also present in samples where you have a quite faint ring that doesn't seem to be saturated, my guess would be that the uneven protein loading is the cause. You can easily test that by staining the membrane with Ponceau S after drying the membrane. Uneven loading can result from concentration of the protein at the borders of the spot, if you just apply a drop onto the dry membrane. The best thing to avoid this problem is to use a suction apparatus and higher loading volumes.
Hi. What is the concentration of the protein in the membrane? If you have diluted it 100 times maybe it could be an effect due the pour quantity of protein in the solid phase.,When this occurs the protein tends to go to the border and leaves a white zone between the borders. This effect occurs with all the samples and with all the spots?
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