I have stripped with a harsh stripping reagent such as B-mercapto ethanol but I lost most of the protein
Have you tried Abcam mild stripping buffer?? I use it on PVDF and works well. http://www.abcam.com/ps/pdf/protocols/Stripping%20for%20reprobing.pdf
Have you tried Abcam mild stripping buffer?? I use it on PVDF and works well. http://www.abcam.com/ps/pdf/protocols/Stripping%20for%20reprobing.pdf
This is what we use for stipping membranes: for 100ml: 20ml SDS 10%, 6,25 ml Tris HCl pH6,8 , 0,7 ml mercaptoethanol, water.
We leave the membrane during 30 min at 60°C, or 45°C if the protein does not stand the treatment.
0.5M NaOH for 15 ml at RT (25ml per membrane) on shaker. Followed by 2X washes in TBS-T at RT on shaker (5 mins ea) and 1X wash in TBS ( 5 min) at RT on shaker. It is for PVDF
Before anything, always dry your pvdf membranes after transfer (as suggested by Lambros Panagis: briefly rinse in dd. water and then dry them completely). Proteins will stay better on membrane, so any kind of stripping will not remove valuable samples you wish to probe. Otherwise, all stripping procedures suggested are good and working. Please note that NaOH treatment will rather erase HRP than remove antibodies. If you want complete stripping, either go the hard and smelly way or use acidic glycine with a pinch of SDS (as suggested by Diego, Abcam provides protocols for this). Good luck
Can I use these protocols for nitrocellulose membrane? We try with NaOH 0.8M but some proteins lost.
Anyone knows if I can use the 25 mM glycine pH 2.0 + 1-2% SDS protocol for use with nitrocellulosa membranes??
This protocol was written by Hassiba Beldjoud
Keep blot in an open box and add 0.5 mM EDTA (covering whole blot and in excess amount) . Place your box in 98 degree water for 5 min or put in microwave oven for 1.5 min. A nice stripping without loss of any protein. Good luck.
I've been succesfully using abcam's mild buffer recipe (200 mM glycine, 0.1% SDS, 1% Tween20) in PVDF membranes for 2 years. It's quite gentle with the proteins in the membrane although it doesn't strip completely the antibodies when facing very abundant proteins.
Yes this mild stripping buffer works nicely with nitrocellulose as well.
As SV Singh suggest, I used 0.5mM EDTA and microwave for 5 min followed by blocking and reprobing. However the stripping was not found to be good, I still see the previous band (before stripping). Can anyone suggest me to overcome this and Is there any way to confirm the stripping has been done before we reprobe it?
If you are using Nitrocellulose then 0.2N NaOH for 15 mins will do the job very well. If you are using PVDF Use this Buffer
Stripping buffer: 62.5 mM Tris pH 6.8, 2% SDS, 100 mM BME
20% SDS (x) = 75 mL (2%) 7.5 mL
2M Tris (x) = 75 mL (62.5 mM) 2.343 mL
500 ul BME/ 50mL
64.587 mL H20
Just incubate blot in this buffer? For how long and what temperature. Thanks
You will have to try it for different durations. It depends on the quality of the antibody and how strong the antibody (either primary or secondary) bind to the protein. I generally start with 10 minutes and the maximum I have incubated is 30 min at RT. Since SDS is a strong denaturing agent you don't have bother about temperature.
Hi,
You add 25 ml stripping buffer to the membrane and incubate in 50°C for 30 min.
Remove the stripping buffer and add blocking buffer (5%). Incubate for 1h at RT (or in 4°C over night).
Now you can add your primary ab and continue as usual.
Stripping buffer:
30 ml Tris HCL pH 6,6
50 ml 20% SDS
416,5 ml dH2O
Add 350 ul β-MeOH per 50 ml
I have to strip a PVDF membrane but i need to do it the day after i detect it, i was thinking to keep it at 4° O/N with TBS. Have some of you ever done this before?
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