The NewBlot IR and WesternSure ECL Stripping Buffers will demonstrate decreased performance when used at 2X or 5X concentration and heating the solution will not increase the stripping efficiency. Performance of NewBlot Nitro and PVDF Stripping Buffers may be improved by optimizing buffer concentration, temperature and time. See the Optimization sections below for detailed instructions.
For NewBlot IR and WesternSure ECL Stripping Buffers, we recommend loading a maximum of 30 µg to prevent membrane saturation with target antigen. Amounts greater than this will decrease the effectiveness of the stripping buffer.
Typically, up to three times. The number of times is dependent on several factors, including the type and amount of bound antigen, the type of membrane used, and the stringency of stripping conditions.
Blotted target proteins can be lost from the membrane during stripping. Protein loss is likely to be influenced by amino acid composition, so different target proteins may be affected differently. Quantification can be performed to evaluate band intensities within the reprobed blot, but not to compare band intensities from previous analyses of the same blot. Only qualitative analysis should be performed when comparing stripped/reprobed blots.
Freshly-prepared stripping solution should always be used. The presence of infrared dyes in previously-used stripping solution will contribute to high background fluorescence when the blot is reprobed.
Typically, reblocking the membrane after stripping is not necessary. If you see an excessive amount of background signal, however, additional blocking may help. Block for 15-30 minutes in Blocking Buffer prior to reprobing your membrane.
There are many factors that affect the overall performance of the NewBlot Stripping Buffer. Please refer to the pack insert for the specific stripping buffer that you are using for optimization guidelines.
NewBlot Stripping Buffer has been used with a number of fluorescently-labeled antibodies from other manufacturers, and has worked quite well in most cases; however, NewBlot Stripping Buffer is optimized for use with LI-COR IRDye 800CW, IRDye 680LT, and IRDye 680RD secondary antibodies. NewBlot Nitro Stripping Buffer cannot be used with IRDye 700DX secondary antibodies.