Please look at the attached photo. I have tried to develop MMP-3 band in HL-60 and HCT-15 cells. Usually i get the dark black backgroung, although i do the washing for about 1 h with 5 and 10 minutes time interval, using TBST. I also block the NC membrane with 5% non-fat milk for 2 hours at 4°C. Primary antibody conc. I use is: 1:1000 and it is 1:2000 for secondary antibody. I use ECL reagent by Amersham, and i put it on the NC membrane in the quantity to wet it fully, and access ECL reagent is wiped with tissue paper. I get the bands on X-ray, but due to dark backgroung, i can't use it. Please share your views with me. Thanks
Dear Salman,
to our experience it is good to try (as suggested above) to block your blot at room temperature. Make sure you are using TBST at the correct pH. Also, I am not sure about your antibody, but 1:2000 secondary seems to be excessive. You could try 1:5000 or even 1:10000 (this is out typical dilution).
Best wishes,
Nikolas
you need to block on room temperature, the black background shows non-specific proteins.
Blocking step should be optimized to correct the dark background.
Dear Salman,
to our experience it is good to try (as suggested above) to block your blot at room temperature. Make sure you are using TBST at the correct pH. Also, I am not sure about your antibody, but 1:2000 secondary seems to be excessive. You could try 1:5000 or even 1:10000 (this is out typical dilution).
Best wishes,
Nikolas
I wonder if it is only a matter of blocking, since the space in between the lanes is not black...Maybe you have a SDS-PAGE problem as well? What kind of gel do you use?Did you try loading different concentrations of your lysate? Did you do a protein stain (Coomassie) on a parallel gel? How does it look like?
Concerning blocking and antibody concentration I agree to the others. Sometimes Tween in the blocking buffer gives you nonsense background - you might try different buffers in the blocking step (PBS, TBS, PBST, TBST).
You may need to check if the detection system you are using is biotin/avidin. If this is the case, then using nonfat powdered milk as a blocking agent might be causing the background. You should use bovine serum albumin instead.
Also, you may want to check your membrane washes and the washing buffer/solution.
Western blot background can include uneven splotchy background, random black smudges, white spots or cleared areas, uneven bands, and random speckles. There are several reasons for this type of background.
a. Dirt
Chemiluminescent reagents are attracted to dust and dirt that may fall onto the blot during washes or be transferred to the blot from dirty equipment. Background from dust and dirt is usually seen as random speckles on the blot.
To prevent dust and dirt contamination, wash all equipment that comes in contact with the membrane, this includes the transfer apparatus and trays used for incubations. Filter all buffers and solutions to remove particles. Keep covers on dishes to prevent dust from falling into solutions during incubations.
b. Poor Handling of Membrane
Splotches, lines and dark spots often arise due to poor handling of membrane. Thus, always handle membranes with gloved hands and use forceps for manipulations. Oils on the hand will cause dark splotches. Be careful not to fold the membrane making a crease, this will cause dark lines. Do not scratch the membrane when adding solutions or using forceps. Make sure the membrane is saturated with solutions during incubations to prevent areas from drying out.
c) Buffer components, such as detergents and blocking agents can stick to the membrane and cause dark spots if they are not completely solubilized. This is particularly common with non-fat dry milk. So, completely mix buffer components
Make sure all solutions are mixed thoroughly before use; blocking solutions can be vortexed. Solutions can also be filtered before use.
From your picture it looks just the specimens lines have aspecific binding. But it could also be you are exposing the membrane for too long.
I usually block the membrane for 1h @ room temp. with 5%milk/TBST (but you can leave the membrane in blocking overnight). Depending on the antibody I do Primary for for 2h @RT or overnight @4C. Concentration is 1/1000 in blocking buffer(i.e with milk), corresponding to 1ug/ml of antibody.
Secondary is always 1:2000 for 30min @RT
Of course you need to make sure you are handling the membrane making sure it doesn't get dirt from anywhere (hands or instruments)
Hi Salman,
Since there are clean spaces between the top of each lane, it looks like you may have simply exposed your film for too long. This is especially likely if your protein of interest is highly expressed. The Amersham ECL kit is also very sensitive.
If you haven't yet, I would do a very quick exposure for less than a second. I've had good luck smacking the film onto the membrane and immediately removing it such that the x-ray film was only exposed for a fraction of a second. Good luck!
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