Ghost bands in western blot - Western blotting problems (Jun/19/2009 )

Hi,

I have been working on a western blot for a protein for months now and almost losing my sanity. I have used the same antibody and different preparations of protein lysates. My most recent problem is the appearance of ghost bands (thick white bands almost like blotches). I had encountered a similar problem in the past but the white bands appeared only in the lanes where the protein was known to be expressed in higher amounts. However, recently all of my lanes have proteins from the same type of cells. The bands appear around the region of my expected protein band. There are othe dark/sharp bands present above and below this region. After googling hours for answers, my conclusion is it could be coz of high secondary antibody concentration (I used 1:10,000). I have no idea if this membrane can be salvaged or I need to repeat this altogether. I would really appreciate if someone would suggest what I can do to prevent this.


-Distressed Grad Student

pm1234 on Jun 19 2009, 07:55 AM said:

Hi,

I have been working on a western blot for a protein for months now and almost losing my sanity. I have used the same antibody and different preparations of protein lysates. My most recent problem is the appearance of ghost bands (thick white bands almost like blotches). I had encountered a similar problem in the past but the white bands appeared only in the lanes where the protein was known to be expressed in higher amounts. However, recently all of my lanes have proteins from the same type of cells. The bands appear around the region of my expected protein band. There are othe dark/sharp bands present above and below this region. After googling hours for answers, my conclusion is it could be coz of high secondary antibody concentration (I used 1:10,000). I have no idea if this membrane can be salvaged or I need to repeat this altogether. I would really appreciate if someone would suggest what I can do to prevent this.


-Distressed Grad Student

Is your first antibody very specific?
If it's not too specific you might detect some chaperone which are often upregulated during protein overexpression and can make up about 1/3 of total cellular protein...

For best results, run the gel again with less sample per well, use at least 5 fold less primary antibody and expose for lesser time. The white bands just mean ur signal is saturated- too much protein- too good and too much primary and too much secondary antibody. In this case, short exposure will not help unless u can do microseconds- I didn't think so

one of my colleagues had this problem and our supervisor told her to just develop the film after 1 second. It worked, but not as well as it should.

Try rerunning the samples with lesser proteins. Dilute the proteins and keep everything the same, this should solve the problem. Your main purpose is to see if the protein is there, right?

Thanks everyone for your suggestion. I will repeat will less sample and more dilute antibodies this time. I am using ECL reagent from pierce (super signal west femto) and I am using a kodak imager to take the pictures.

My western is for some knockout experiments so I will have to quantify my protein expression not only see if its there or not.

the white ghosts bands are the result of quenched ECL substrate. make sure you dont wait too long after rinsing your membranes with ECL solutions AND not to overload the protein of interest.

there are 2 major types of ECL solutions, the normal ones are best suited when u have ample amount of protein on the membrane to detect. but if have your membrane overloaded with protein the HRP will use up all of the substrate resulting a no reaction region(ghost band) in your film ASAP.
the enhanced ECL solutions are best suited when ur protein of interest is in negligible amounts. even here if you wait too long after rinsing your membranes, the ECL reactions are quenched resulting no signal.

Any update in what happened? I am having the same problem. I load 10ug of protein, 1/700 primary antibody ovrn 4C, 1/10,000 2ry 1hr. I have done it like that before and it has worked, and now I don't know why I get these ghost bands. If I expose a minute nothing will show up, if I expose like 10min then I see the ghost bands.

I found this on the BioTechniques website:
"If the primary antibody concentration is too high and blocking is not sufficient, some primary antibody may bind to places there is no protein transfered. In other words, the bands of protein prevent nonspecific binding of the antibody to the membrane. Subsequent over-development of the blot will show a dark dark background with the bands in "negative image"."

If this is the case, can I take the membrane and reblock it and then add more secondary ab?