use of Pierce stripping buffer in WB - (Jan/22/2007 )
Hi,
I just started working with western blot. In WB, it is necessary to strip the membrane and reprobe it. I found this step is tricky to me. I used the stripping buffer from Pierce. The manufacturer's manual recommends 5-15min incubation of the membrane at room temperature, and of course, optimization is required. To facilitate optimization of incubation time and temperature, the manufacture recommends two ways to test: 1) test the removal of the secondary antibody by incubating the membrane with developing reagent after stripping, followed by exposure to films; 2) test the removal of the primary antibody by incubating the membrane with secondary antibody, then developing and exposure. When I used the stripping buffer (15min at room temperature), I then did test 1 and confirmed that the secondary antibody has been removed. However, when I then did test 2 (after washing once in PBST), I saw more non-specific bands on the film than before stripping. I don't know how to interpret the result. Should I extend or reduce the incubation time in the stripping buffer? I would really appreciate it if anyone who has experience with this product can give me any advice.
I just started working with western blot. In WB, it is necessary to strip the membrane and reprobe it. I found this step is tricky to me. I used the stripping buffer from Pierce. The manufacturer's manual recommends 5-15min incubation of the membrane at room temperature, and of course, optimization is required. To facilitate optimization of incubation time and temperature, the manufacture recommends two ways to test: 1) test the removal of the secondary antibody by incubating the membrane with developing reagent after stripping, followed by exposure to films; 2) test the removal of the primary antibody by incubating the membrane with secondary antibody, then developing and exposure. When I used the stripping buffer (15min at room temperature), I then did test 1 and confirmed that the secondary antibody has been removed. However, when I then did test 2 (after washing once in PBST), I saw more non-specific bands on the film than before stripping. I don't know how to interpret the result. Should I extend or reduce the incubation time in the stripping buffer? I would really appreciate it if anyone who has experience with this product can give me any advice.
The Restore stripping sol'n. (I assume this is the product you're referring to) seems to work well with low affinity antibodies; however, with better antibodies (i.e. antibodies with a high affinity for the target antigen), the Restore solution is inadequate. Instead I use a mercaptoethanol stripping solution. PM me if you want my protocol or have any other questions. Also, what kind of membrane are you using?
I was at the Pierce site yesterday when I noticed a new stripping solution: Restore Plus Stripping solution. Depending on the reason for reprobing, another option for you may be to cut the blot such that antigen 1 is on one piece and antigen 2 is on the other and then process the two portions in parallel with their respective antibodies.
Good Luck
Maybe you forgot to block again after stripping?
sometime also membranes are slightly "damaged", and one can see more background, so you might optimize your blocking.
On the other hand, Mondo is right : I was not always able to strip all my antibodies, depending on their affinity, so I stripped in :
-345 µL beta mercaptoethanol
-10 mL SDS10%
-2.5 mL tris HCl pH 6.8 1M
-water up to 50 mL
incubate 30 minutes at 50°C
we use Pierce stripping solution longer than the recommended 15 min; we wash then in TTBS but do not block again; stripping is more efficient for 2D blots than for 1D blots; we re-probe 2D blots several times without increased unspecific background; high conc of secondary Ab may result in a lot of unspecific bands; may be your 2nd Ab titer was too high or too long exposed to autoluminescence