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Protein Stripping Buffers - Category Selection Guide

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layers_clear Low-pH · Detergent Stripping Chemistries

Protein Stripping Buffers - Category Selection Guide

Two chemistries, one page. Pick the stripping route your blot can tolerate and go straight to its product page — the mechanism, the history, and the re-probing guidance follow below.

Protein Stripping Buffers — Catalog · 2 Chemistries
Select the chemistry that matches your membrane and antibody — click View for the product page.
Stripping Buffer Cat. No. Best Use Product Page
Glycine Low pH Antibody Stripping Buffer DCP-GLPHASB1X Gentle standard for multiple re-probes — acidic disruption of antibody binding at room temperature; minimizes target loss, favored for nitrocellulose and sequential re-probing Viewarrow_forward
SDS Stripping Buffer DCP-SDSSB1X High-affinity antibodies — strong detachment power for resilient primary / secondary complexes; effective on PVDF where robust stripping is needed Viewarrow_forward

Not sure which chemistry? See what a stripping buffer has to deliver · See applications by workflow · Read the FAQ

Category Snapshot

Getting more than one answer out of one membrane

Western blot samples are often precious and limited, and running a fresh gel for every target is slow and expensive. Protein stripping buffers remove primary and secondary antibodies from a nitrocellulose or PVDF membrane without releasing the underlying immobilized proteins, allowing researchers to re-probe the same membrane for multiple targets.

The chemistry that decides how much signal you keep — and the limitation that decides how you are allowed to read the result.

  • Two chemistries stocked: Glycine Low pH Antibody Stripping Buffer and SDS Stripping Buffer.
  • What is actually being undone: antibody–antigen binding relies on hydrogen bonds, hydrophobic contacts, and electrostatic forces. A good stripping buffer undoes these selectively.
  • Selective dissociation shifts the molecular environment enough to release the detection antibodies while leaving target proteins bound to the hydrophobic membrane.
  • Conserved sample value: multi-target analysis — for example a target protein plus a loading control — on one blot, removing gel-to-gel loading variation.
  • Time and cost efficiency: reusing a single membrane reduces transfer time and reduces consumption of gels, transfer reagents, and membranes.
  • The limitation is real, not a formality: every stripping cycle removes some immobilized protein, and most antigens tolerate only a limited number of cycles.
  • Read re-probed blots accordingly: they are best treated as qualitative / confirmatory rather than strictly quantitative.
Western Blot Re-Probing RUO
CATEGORY REFERENCE · PROTEIN STRIPPING BUFFERS
Stripping selection at a glance — chemistry, conditions, and what each route costs you
  • Products in this category2
  • Glycine route pH~2.0–2.5
  • Glycine route temperatureroom temperature
  • SDS route mechanismdetergent-driven denaturing system
  • Original 1987 protocol componentsSDS + β-mercaptoethanol + heat
  • Original 1987 protocol heat~50–70 °C
  • Membranes namednitrocellulose, PVDF
  • Multi-antibody probing first described1981
  • First widely used stripping protocol1987
  • Cycles allowed by the harsh early methodone or two
Why Protein Stripping Buffers Are Needed

Release the antibody, keep the protein

Stripping is a selectivity problem, not a strength problem. Each panel below covers one half of it — what the buffer has to break, and what it has to leave alone.

link

What holds the antibody on

Antibody–antigen binding relies on hydrogen bonds, hydrophobic contacts, and electrostatic forces. A stripping buffer has to undo that set of interactions on purpose rather than by brute force.

filter_alt

Selective dissociation

A good stripping buffer shifts the molecular environment enough to release the detection antibodies while leaving target proteins bound to the hydrophobic membrane.

savings

Conserved sample value

It enables multi-target analysis — for example a target protein plus a loading control — on one blot, removing gel-to-gel loading variation. Western blot samples are often precious and limited.

schedule

Time and cost efficiency

Reusing a single membrane reduces transfer time and reduces consumption of gels, transfer reagents, and membranes. Running a fresh gel for every target is slow and expensive.

trending_down

Every cycle costs you protein

Every stripping cycle removes some immobilized protein, and most antigens tolerate only a limited number of cycles. This is a real limitation, not a formality.

rule

Read a re-probed blot correctly

Re-probed blots are best treated as qualitative / confirmatory rather than strictly quantitative. Plan the probing order so the reading that matters most comes off the membrane first.

The trade-off that decides the chemistry

Detachment power and target retention pull against each other. The SDS route is a detergent-driven denaturing system with strong detachment power for resilient primary / secondary complexes — more aggressive, so watch target-protein loss. The glycine route disrupts antibody binding acidically at room temperature and minimizes target loss, which is what makes it the gentle standard for multiple re-probes. Neither escapes the caveat: some immobilized protein leaves with every cycle.

1987
the first widely used stripping protocol — effective but harsh, limiting a membrane to one or two cycles
2.0–2.5
pH — the low-pH glycine route, working at room temperature
History of Protein Stripping Buffers

From erasing a blot to re-probing it repeatedly

Three developments moved re-probing from a proof of concept, through a harsh but working protocol, to the gentle commercial buffers stocked today.

  1. 1

    1981 The multiple immunoreplica technique

    Legocki & Verma described the “multiple immunoreplica technique” for probing one gel / membrane with a series of different antibodies. It was an early demonstration that a single immobilized-protein sheet could be interrogated for multiple targets — the conceptual root of re-probing.

  2. 2

    1987 The erasable Western blot

    Kaufmann, Ewing & Shaper published “The erasable Western blot,” using detergent (SDS), a reducing agent (β-mercaptoethanol), and heat (~50–70 °C) to detach antibodies. This established the first widely used stripping protocol — effective but harsh, often removing target protein too and limiting a membrane to one or two cycles.

  3. 3

    1990s–2000s Gentler methods

    Low-pH (glycine) buffers and optimized detergent systems emerged that disrupt antibody–antigen binding at room temperature, without heat or odorous reducing agents. These enabled modern commercial stripping buffers offering rapid, repeatable re-probing with far less target-protein loss.

Requirement Reference

What a stripping buffer has to deliver

Three requirements, and the caveat that governs all three. Use this table to translate a re-probing plan into the chemistry that supports it.

Requirement How the buffer delivers it What you get back
Selective dissociation Shifts the molecular environment enough to release the detection antibodies Target proteins stay bound to the hydrophobic membrane
Conserved sample value Enables multi-target analysis on one blot — for example a target protein plus a loading control Removes gel-to-gel loading variation
Time and cost efficiency Reusing a single membrane reduces transfer time Reduces consumption of gels, transfer reagents, and membranes
Important caveat. Every stripping cycle removes some immobilized protein. Re-probed blots are best treated as qualitative / confirmatory rather than strictly quantitative, and most antigens tolerate only a limited number of cycles. This is a real limitation, not a formality.
On the 1987 conditions. The source gives the historic method as detergent, reducing agent, and heat at approximately 50–70 °C. As published, the protocol itself specifies 2 % SDS with 100 mM 2-mercaptoethanol for 30 minutes at 70 °C; the wider range in general circulation reflects later variations on it. Nothing in the source is wrong here — it is worth knowing which end of the range the original sits at before a legacy protocol is copied into a new SOP.
Applications

Which re-probing job, which chemistry

Select a workflow to see the application the source assigns to it, and the stocked chemistries that serve it.

Loading-control normalization · one blot, two readings
  • Re-probing for housekeeping proteins (GAPDH, actin, tubulin) after detecting a target, to confirm equal loading
  • Running the target and its loading control on one blot removes gel-to-gel loading variation
  • Glycine Low-pH Stripping Buffer — the gentle standard for multiple re-probes; minimizes target loss
Phosphorylation-state analysis · activation ratios
  • Detecting a phospho-specific form, stripping, then re-probing for total protein to calculate activation ratios
  • Glycine Low-pH Stripping Buffer — acidic disruption of antibody binding at room temperature, favored for sequential re-probing
  • SDS Stripping Buffer — strong detachment power where the phospho-specific antibody is high-affinity and resists a gentler route
Multiplex sequential analysis · scarce sample
  • Screening one sample against multiple distinct-molecular-weight targets when sample is scarce
  • SDS Stripping Buffer — detergent-driven denaturing system; effective on PVDF where robust stripping is needed
  • Bear in mind that most antigens tolerate only a limited number of cycles
Product Comparison

Core mechanism and best use, side by side

The source’s product comparison, reproduced row-for-row. Product labels are the source’s own.

Product Core Mechanism Best Use
SDS Stripping Buffer Detergent-driven denaturing system High-affinity antibodies — strong detachment power for resilient primary / secondary complexes; effective on PVDF where robust stripping is needed. (More aggressive, so watch target-protein loss.)
Glycine Low-pH Stripping Buffer Low pH (~2.0–2.5) dissociation Gentle standard for multiple re-probes — acidic disruption of antibody binding at room temperature; minimizes target loss, favored for nitrocellulose and sequential re-probing
Label note. The catalog above lists the glycine product under its catalog name, Glycine Low pH Antibody Stripping Buffer (DCP-GLPHASB1X); this table uses the source’s own label, Glycine Low-pH Stripping Buffer. Both are reproduced as published and neither was edited.
FAQ

Frequently asked questions

The questions that come up most often when a re-probing plan meets a purchasing spec.

It removes primary and secondary antibodies from a nitrocellulose or PVDF membrane without releasing the underlying immobilized proteins, so the same membrane can be re-probed for multiple targets. Antibody–antigen binding relies on hydrogen bonds, hydrophobic contacts, and electrostatic forces; the buffer shifts the molecular environment enough to release the detection antibodies while leaving target proteins bound to the hydrophobic membrane.
The glycine low-pH buffer is the gentle standard for multiple re-probes: acidic disruption of antibody binding at room temperature that minimizes target loss, favored for nitrocellulose and sequential re-probing. The SDS buffer is a detergent-driven denaturing system for high-affinity antibodies, with strong detachment power for resilient primary / secondary complexes — more aggressive, so watch target-protein loss.
It is part of the choice. The SDS buffer is described as effective on PVDF, where robust stripping is needed. The glycine low-pH buffer is favored for nitrocellulose and for sequential re-probing. Both chemistries address blots on nitrocellulose or PVDF membranes.
Most antigens tolerate only a limited number of cycles, because every stripping cycle removes some immobilized protein. The harsh original protocol of 1987 often removed target protein too and limited a membrane to one or two cycles; the gentler modern buffers were developed precisely to allow rapid, repeatable re-probing with far less target-protein loss.
Re-probed blots are best treated as qualitative / confirmatory rather than strictly quantitative. Some immobilized protein is lost on every cycle, so the signal from a later probe is not on the same footing as the first. This is a real limitation, not a formality — plan the probing order so that the reading which matters most is taken first.
Two reasons. Sample value: Western blot samples are often precious and limited, and running one blot for a target protein plus a loading control removes gel-to-gel loading variation. Cost and time: reusing a single membrane reduces transfer time and reduces consumption of gels, transfer reagents, and membranes, where running a fresh gel for every target is slow and expensive.
Nothing, for its time — Kaufmann, Ewing and Shaper’s “erasable Western blot” established the first widely used stripping protocol, using detergent (SDS), a reducing agent (beta-mercaptoethanol) and heat at roughly 50–70 °C. It was effective but harsh: it often removed target protein too, and limited a membrane to one or two cycles. Low-pH glycine buffers and optimized detergent systems developed through the 1990s and 2000s work at room temperature without heat or odorous reducing agents.
Key References

The primary literature behind these methods

The source’s reference list, reproduced and re-verified against the primary literature. DOIs are shown only where a real one exists.

  1. Legocki, R. P., & Verma, D. P. S. (1981). Multiple immunoreplica technique: screening for specific proteins with a series of different antibodies using one polyacrylamide gel. Analytical Biochemistry, 111(2), 385–392. — An early method for interrogating one immobilized-protein sheet with multiple antibodies. doi:10.1016/0003-2697(81)90577-7
  2. Kaufmann, S. H., Ewing, C. M., & Shaper, J. H. (1987). The erasable Western blot. Analytical Biochemistry, 161(1), 89–95. — The foundational stripping-and-reprobing protocol (detergent / reducing agent / heat). doi:10.1016/0003-2697(87)90656-7
  3. Alegria-Schaffer, A., Lodge, A., & Vattem, K. (2009). Performing and optimizing Western blots with an emphasis on chemiluminescent detection. Methods in Enzymology, 463, 573–599. — A comprehensive modern guide to Western blotting practice, including stripping and re-probing. doi:10.1016/S0076-6879(09)63033-0
Stripping selection support. For help matching a stripping chemistry to a specific antibody, membrane, or re-probing sequence, or for documentation requests, contact support@diagnocine.com. Ready to order? Back to the Protein Stripping Buffers catalog.

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