Due to unavoidable circumstances the PVDF membrane was kept in ponceau stain for 20 minutes forcing us to destain it using Milli-Q water for 1 hour.
The chemiluminescence detection following it returned no signal.(completely empty blots without even back ground.)Does the above mentioned mistae have something to do with it?
Since Ponceau is a negative stain and the PVDF is electrically neutral, it is reversible. I have never seen an instance when Ponceau is not completely removed. I use nitrocellulose and have left the stain on the blot for 15 minutes with no problem. On abcam's website, they provided the following information. Hope this helps
3. Visualization of proteins in membranes: Ponceau Red
To check for success of transfer, wash the membrane in TBST (for a TBST recipe, see below). Dilute the stock Ponceau Red 1:10. The stock is made of 2% Ponceau S in 30% trichloroacetic acid and 30% sulfosalicylic acid.
Incubate on an agitator for 5 min.
Wash extensively in water until the water is clear and the protein bands are well-defined.
The membrane may be destained completely by repeated washing in TBST or water. When using a PVDF membrane, re-activate the membrane with methanol then wash again in TBST.
Thank you , Mr. Kemp. Could you please elaborate on re-activating a PVDF membrane because I do activate my membrane prior to transfer using a semi-dry blotting apparatus.
Apart from that can the acetic acid in Ponceau stain have any detrimental effects?
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