In the images I have loaded decreasing concentrations of protein, I have also done tests with different dilutions of antibody. The reference antibody comes out very clean, whereas the antibody of interest cannot get a good image. Can anyone give me any ideas?
hi
you can check the positive control (such as MCF-7 cell line ) 20 ug/well with different concentration of primary antibody .After getting the right result, then run the original test.
"If searching GATA3 antigen = 50kD"
good luck
Hi Ana Salinas,
If I were you I would try two different things in two different runs:
1- Load higher amounts of lysate per each well. You can start with ~ 100 ug/well. This suggestion is based on the probability that your protein of interest is lowly expressed in the tested lysate compared to the positive control.
N.B. The intensity of the band of your positive control will be very much increased during detection, but it`s a positive control so no worries!
2- Use higher concentrations of your primary antibody. In other words, use a less diluted one. Check the literature for this purpose, Don't rely totally on the datasheet.
Also please, think about using a more sensitive detection system:
- If you are using Pico, you can switch to Femto substrate
- X-ray based system, though it is old fashioned, is more preferred to digital camera-based systems.
You may also consider changing the blocking and antibody dilution buffers:
- If you are using BSA, switch to 5% skim milk and vice versa.
Your membrane looks with some background, please, use fresh buffers.
Since one of your antibodies work well, I guess there is nothing wrong with your methodology. Indeed, your primary antibody for your target gene has probably a low quality and maybe it is polyclonal. Monoclonal antibodies from outstanding companies (e.g. Abcam, Roche) are the best choices. However, if not possible to change your antibodies, you should try elongating the incubation period with blocking buffer. Shortening the exposure time is another way that may work for eliminating the unspecific bands and backgrounds.
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