Hey Arlind,

Anti His antibody does have its problems, its not as specific as they say it is.

You may try the following to get better signal/noise ratio.

1. I generally don't like skim milk that much because it doesn't always dissolve very well and tends to settle down if kept for longer time- try BSA instead.

2. Did you also use 5% skim milk to make dilution of the anti his antibody? It is a good idea to keep the antibody also in the blocking buffer to minimize nonspecific binding. But since BSA is prefered choice you may also use 5% BSA for the dilution.

3. You may try reducing incubation time with the anti his antibody to 45 min instead of 1 h or more.

4. You may also increase the dilution from 1:2000 to 1:4000.

5. And most of all try to make sure that you have a good signal in your sample to begin with so, I would use a positive control to see if the conditions I am trying is working as desired. I believe for colorimetry 200 ng positive control will give good signal.

Good luck,

S

Try an unconjugated primary Ab and then a secondary conjugated to HRP. Wash the blot using large volumes.

You could also try a chemiluminescence substrate instead of DAB with a short exposure time.

A couple of hints to make your blots work better:

• Use PVDF, not nitrocellulose, the protein binding capacity and affinity are higher and the membranes do not fragment during the many incubation steps.
• Your use of a tank blotter is good, they work better than semi-dry. You can reduce the voltage somewhat, 30-40 V for 1 h should do. That keeps the heat down. I use Dunn's blotting buffer (10 mM NaHCO3, 3 mM Na2CO3, doi:10.1016/0003-2697(86)90207-1), its cheaper and better than Towbin's.
• From my personal experience I'd recommend Rainbow markers (originally Pharmacia, then Amersham, then GE Healthcare, now Cytiva). Each band has a different colour, making identification easy. And I have never lost colour from my blots.
• Do not block the membrane for too long, 15 min at RT are sufficient. Otherwise, a layer can form on top of the blotted bands and reduce sensitivity.
• I'd dilute the antibody more, this might improve specificity. Also know that not all antibodies were created equal. I had good results with Accurate and Dako.
• If you add 1 mM Ni2+ to your DAB-reagent, the precipitate from the HRP reaction reduces it to black Ni0, which is stable in your notebook and easy to photograph.

Btw, I have no affiliation with any of the companies mentioned.

Arlind Mara

I can make out some clear points by looking at the photographs of western blot:

1. You need to optimize the load..... doesn't seem to appropriate to me at all.

2. to optimize the dilutions for anti-his.

3. If this is your first one ... then please try again to make sure that you follow everything according to the protocol.

4. I clearly see that there is also a handling problem of membrane.

I wish you best !!

Any pair of molecules (Ab-Ag) will have some degree of affinity; the more abundant the epitope the more likely you'll pick up a signal with ANY Ab. If you can ponceau stain your bands, they are quite abundant and will be recognized some Abs non-specifically. HIS Abs are noteworthy for promiscuous binding. I would suggest you repeat your WB expt with considerably less material (e.g. 20x-100 x less) to minimize off-target binding by your Ab. You should have ng range (pg for some) not ug range concentrations of you epitope, at least for Western blotting. Good luck!