Stripping a blot and why it can fail

Stripping a blot means removing the antibodies from a membrane so a second probe can use the same sheet. It fails in two opposite directions. A harsh strip can lift the antigen off with the antibody, so the second probe has less protein to find. A mild strip can leave the first signal in place, so the second film still shows the first target. This page is a decision guide for stripping or rerunning. The blot you are considering stripping was made as in western blot from gel to membrane. Whether the second primary is a fair reagent is choosing and checking a primary antibody. The photograph above is not the membrane. It is an agarose DNA gel under an orange ultraviolet shield, the kind of record described in agarose gel electrophoresis for DNA.

Membranes and detection reagents are enquiry classes in the molecular biology catalogue. Say whether you plan to reprobe on the quote request.

When a strip is the wrong first idea

Cut the membrane if the two masses sit far apart and you marked the sheet, with the ladder, before you sliced. Use two fluorescent channels if the primaries are from different hosts and your imager can keep the colours apart. Run a second gel if the claim is quantitative and you have lysate left. Stripping is what remains when the signals occupy the same undivided sheet and you cannot detect them together.

A loading control is the usual reason people strip, and a common way the strip misleads. A housekeeping band is not automatically stable across the treatment. A total-protein stain made on the transfer day, before any antibody, never needs a strip. If that picture already exists, do not strip the blot merely to add actin.

What mild and harsh strips do

A mild strip is usually a low-pH glycine class buffer, sometimes with a little detergent, used at room temperature for a short time. It is meant to release antibody and leave the polypeptide on the sheet. High-affinity antibodies and enzyme conjugates often survive it. The first band then remains, and the second probe is read on top of a ghost.

A harsh strip adds stronger detergent, often a reducing agent, and often heat. It is more likely to clear the first signal. It is also more likely to pull antigen off, especially after a second or third round. PVDF is the sheet people usually choose when they plan to reprobe, because it is mechanically tougher. Nitrocellulose can be stripped and becomes fragile. Neither polymer promises that the antigen amount is unchanged.

Re-block after a successful strip. The strip removes part of the original block, and a bare sheet plus a new antibody is a high-background blot. The molecular weight ladder's prestained colours may fade. Do not restitch a size estimate from memory. UniProt still holds the sequence mass. The ladder that remains visible on that sheet is the only ruler left.

Order of probing

Probe the scarcer signal first. An abundant protein is the one more likely to remain detectable after the sheet has been stressed. Probing the loading control first, stripping, and then looking for a rare phosphoprotein spends the rare epitope on a harsher history. Record each exposure before the next strip so a reviewer can see the order.

If the two claims must both be quantitative, do not stack them on one stripped sheet and call them paired. Paired means the same transfer, minimally disturbed. Two channels, a cut membrane, or two fresh blots from the same lysates are the cleaner designs. A strip is a convenience with a chemical cost.

A path you can audit

Save the film or the file of the first probe, including an exposure that is not saturated. Incubate in the strip buffer class you chose, with the time and temperature written down. Wash thoroughly. Add detection reagent without a new antibody and expose for at least as long as the exposure that showed the first band. A blank frame is the permit to re-block and add the next primary. A remaining band withdraws that permit.

Then probe, wash and expose as you did the first time. Compare lane shape with the original file. A second signal that appears in lanes that were empty, or that vanishes from lanes that were loaded, is not a biological story until the detection-only frame was clean. If you cannot produce that frame, rerun.

Safety and the limits of a reprobed sheet

Strip buffers are acidic, strongly reducing, or both, and heated buffer is a burn hazard. Follow the membrane maker's note and the laboratory's chemical rules. Do not invent a stronger mix because the first band was stubborn. Methanol already used on PVDF is still flammable. The lysate's biosafety level is unchanged by the strip. The WHO Laboratory biosafety manual, 4th edition is background for institutional practice, not a permit to call a reprobed research blot a diagnostic test.

A clean second probe supports a second presence-and-size observation on a sheet that still holds protein. It supports a fold change only if you have shown the strip did not move the denominator. Most stripped blots should be described more modestly than the first exposure.

Heat and a timer you cannot trust

A harsh protocol written for a stated temperature is harsher if the water bath was already hot from the room, or if the sheet sits in strip buffer while a power cut stops the shaker and the clock. Time the incubation. Pull the sheet when the time ends. An unattended harsh strip is how antigen leaves without a notebook entry. If the building supply is unreliable, prefer a fresh gel over a strip you cannot time. Humidity that dries the edge during the transfer from strip buffer to wash buffer writes a tide mark into the second exposure. Keep the sheet wet, in the same spirit as any blot handling, even though the photograph at the top is a DNA gel and not this tray.

What to put in an enquiry

State the membrane polymer, how many probes you intend, whether detection is chemiluminescent or fluorescent, and whether the targets can be cut apart by mass. Ask for PVDF if reprobing is the plan, and ask the sheet note's position on stripping rather than assuming every western membrane survives it. Ask whether a quotation is possible. The nucleic acid analysis pathway is the neighbouring route for the DNA gel in the photograph. A membrane sold only as "reprobable" has not yet named how many cycles or which strip class the maker tested.