Hi Mathew,

how do you store your sec Ab? Aliqouts frozen at -20, or do you use the same tube all the time? Did you observe this "floating material" in all aliqouts?

If you are using the same tube for sec Ab all the time you should get a new aliqout or oder a new one! Since the HRP is quite stable, it might still be active while your antibody is degraded.

Another point could be a change in FCS. It happend to me, that an endogenous protein simply "disappeared" after the replacement of our usual FCS to a synthetic serum. Proably the composition of that serum (factors present or absent) resulted in a downregulation of my protein of interest. After using the old FCS again this protein was easily detectable again.

More details on your problem would be helpful to give a less speculative advice!

Best,

Kai

What is the source of the primary antibody. Are your primary antibodies, against your protein of interest (polyclonal/monoclonal) or against a specific Tag.

What's the sample type that you are trying to detect.

Though adding 1 microlitre of secondary is high, but still then development of blue colour in the substrate, makes me feel that your secondary antibodies might be active (stiil functional).

Since you have changed the lot of primary antibody, my suggestion would be to perform blotting using higher concentration of primary antibodies (lower dilution).

Hope it works. Please include controls if possible.

Check your buffers. If you doubt precipitation in secondary, use secondary from other sources or higher concentration.

You can stain membrane by ponceau to check transfer, but I doubt whether you can use the same blot after ponceau to perform ECL.

Most likely it is your antibody ( relying of your findings) although washing buffer (TBST/PBST), Blocking buffer which are kept in stock solution can get contaminated as well and cause such difficulties.

making fresh buffers is also recomended.

You can easily check if your secondary antibody is malfunctioning if you use primary antibody as antigen in ELISA or in dot plot assay and see if you secondary will bind to it.

Thank you everyone for your replies.

My secondary ab is stored at 4 C in one tube. That very well may be my issue.

I will also make a fresh stock of buffers. I forgot to mention that I formerly did so, but about 6 months ago I switched to bulk 10X orders.

I will also test my primary antibody using a dot plot assay. These are all very good suggestions. Thank you once again.

-Matt

You can ECL blots after ponceau- I routinely do this.

One way to check whether your protein has been transferred on the blot is by staining the blot with ponceau reagent. It is washable and then you can proceed to the blocking procedure with the same blot. Another issue can be with your antibody as you said that you store it at 4 C in one tube. You should make the aliquots in small eppendorf tubes and then cover them with the aluminium foil and keep them in -20 C. Also the buffers should be freshly made and transfer should be carried out at 4 C. Hope this helps.

Best Regards,

Hi,

Sometimes when we change the primary antibody and get a new lot we see similar problems. Could you check you catalog and lot numbers and validate with the old ones and write to the company who made it. As you have checked most of the steps very carefully, I would suggest to change the primary antibody lot.

hello every one ,

what happen if i add the protein directly to nitrocellulose membrane then block it with 5% milk then add primary then secondary then read it i got black dot on the same area where i add the protein the whole procedure for testing the primary secondary antibody efficiency but then why i get the whole area black shouldn't be specific for the protein of interest only i will upload the picture here :

first membrane with protein 1ry antibody 2ry antibody .

second membrane with protien 2ry antibody .

third membrane with no protien but with 1ry and 2ry antibody .

Mathew Rynes Did you fixed your problem because I'm also facing the same problem!! I have been doing western blots successfully for many months but in my last couple of blots, I couldn't detect any signal but when I checked the loading control, the bands were good!!

Things I tried:

I prepared new dilution for my primary antibody still i couldn't detect any signal.

Tested my ECL substrate with my secondary antibody