Hello, we have been struggling with the lots of background in our wb membranes probed with an anti-Streptavidin-HRP from Thermofisher (Pierce 21134). Samples contained biotinilated proteins. Every time there is some blobs somewhere and so much background that it is hard so see our biotinilated proteins. I attached the same pic with different contrast. Did anyone face the same problem?
All stepts have been performed with PBS 1X and here the protocol:
- After transfer, rinse off membrane for 5 min in PBS
- Block with BSA blocking buffer (1% filtered BSA and 0.2% Triton x-100 in PBS) for 30 min
- incubation with streptavidin antibody 1:2000 dilution ON at 4C
- Rinse off with PBS three times and do ABS blocking (10% adult bovin serum and 1% triton x-100 in PBS) for 5 min
- Rinse off with PBS three times and incubate with PBS for 5 min
- Develop with ECL for 5 min and acquire
I've always used TBST (0.1% Tween) to wash membranes (3 washes for 5 minutes each with light shaking). The addition of the Tween detergent and longer washes might help.
I would also try the streptavidin at higher dilutions in 1 % BSA in case it's saturating the blots.
I have been working with Streptactin-HRP to recognize TwinStrep tagged proteins. I did trials using BSA or milk to block (although they do not recommend using milk to block membranes as skimmed milk contains biotinylated proteins ) and no difference was found comparing these two for my experiments.
What I do after transfer (semi-dry system):
1. Stain with Ponceau to check the transfer and then, wash with 1x TBS-T (0.02% Tween) until dye is removed.
2. Block with a solution of 5% skimmed milk (powder) in 1x TBS-T for 1hour on an orbital shaker.
3. Wash 3 times for 10min each wash with TBS-T (orbital shaker)
4. Incubate the Streptactin-HRP (I was using a dilution of 1:50.000 IBA lifesciences in TBS-T) overnight in the cold room on an orbital shaker.
5. Next morning, wash 3 times with TBS-T (10min each wash) (orbital shaker).
6. Develop by adding ECL solution, incubate for 1 min and acquire image with iBright Thermofisher or X-ray films.
Normally I get very clean membranes with sharp bands, when the amount of the protein of interest was low, I could see some background from the membrane but nothing crazy. Looking at your membrane, there are patches that are very dirty, make sure when washing the membranes to add enough TBS-T to cover the membranes and avoid folding or bending the membrane, treat the membrane carefully and always use tweezers to work with the membrane.
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