Name: Antibody Stripping Solution
Catalog Number: L7710A, 500ml* GV0480, 500ml 5X
* Enough reagent for 3000 cm2 or 200-400 membrane strips or 20-40 standard blots
• Storage: Store at RT (for long storage: +4C°) (L)
No pungent smelling mercaptoethanol saves time
Antibody stripping is performed at room temperature. No need to heat the blots, saves costly sample
Strip antibodies economical in only 15 minutes at room temperature
Blots can be avoided by re-blocking in most cases.

Western Blot Stripping Buffer

Western BLOT stripping buffer is a solution to remove primary and secondary antibodies from the examined western blot membranes. Antibody removal with this buffer can occur under mild conditions (room temperature, 30 min incubation), minimizing the loss of immobilized proteins from the membrane. When using PVDF membranes, the same membrane can be stripped and reprobed 2-5 times. After stripping, the membrane can be re-probed, either with a different concentration of primary antibody or with a completely different primary antibody.

 

Several protocols have been published for antibody stripping from western blots, including low pH, heat and detergents, and the use of chaotropic agents. Three recommended protocols are presented below. The first applies to any chemiluminescent substrate system and uses a combination of detergent and heat to release the antibody. The second is commonly used for applications where the antibody has to be separated from the antigen and employs a lower pH to alter the structure of the antibody in such a way that the binding site is no longer active.

The third protocol uses the ReBlot™Plus Western Blot Recycling Kit that has been specifically designed to isolate antibodies from western blot membranes. Benefits of this approach include

 

Blocking buffer	Blocking agent	Highlights	When to use	Available formats
StartingBlock	Serum and biotin-free single purified protein	Performs well with a wide range of antibodies and antibody combinations. Compatible with streptavidin systems Blocks in less than 15 minutes	Best for med-high abundant proteins or strong antibody affinity High background with current blocking buffer Stripping and reprobing western blots	PBS TBS PBST TBST
Blocker FL	Single purified protein	Blocks excess nonspecific binding sites to help reduce background fluorescence, Works with both nitrocellulose and low fluorescence PVDF Detergent-free Blocks in 15-30 minutes	Fluorescence western blotting Imaging and storage of dry fluorescence blots	10X
Pierce Clear Milk Blocking Buffer	Clarified and stabilized milk proteins	High performance replacement for homemade milk blocking buffers in Western blotting applications Long shelf-life at room temperature	Use when high background seen with Non-fat Milk Fluorescent and chemiluminescent applications	Borate, pH 7.6
SuperBlock	Serum and biotin-free single purified glycoprotein	Protein-based formulation does not contain any immunoglobulins, albumin or endogenous biotin, making it compatible in many situations where traditional blocking agents fail. Biotin-free for use with streptavidin system Blocks in less than 10 minutes	High background with current blocking buffer	PBS TBS PBST TBST
SEA BLOCK Blocking Buffer	Steelhead salmon serum	Useful in detection methods involving mammalian samples. Particularly effective in applications involving fluorescence imaging	Mammalian samples Fluorescence western blotting	PBS
Blocker BSA	Purified bovine serum albumin	10% solutions of high-quality bovine serum albumin Single purified protein provides fewer chances of cross-reaction with assay components than serum or milk solutions	Use when targeting phospho- proteins Best to use when storing reused antibodies in blocker	PBS TBS
Blocker Casein	Purified casein	Single-protein blocking buffer provides fewer chances of cross-reaction with assay components than serum or milk solutions	Use when high background seen with Non-fat milk blockers	PBS TBS
Blocker BLOTTO	Non-fat dry milk	Ready-to-use 5% solution of nonfat powdered milk	Convenience- ready-to-use More consistent product over home-made blockers	TBS
Protein-Free	Non-protein blocking compound	Minimizes or eliminates cross-reactivity associated with protein-based blocking buffers. Sample-and-antibody combinations require the elimination of all possible exogenous animal proteins in the assay system to avoid cross-reaction or quenching of the desired probe function	Use when protein-based blockers cause high background	PBS TBS PBST TBST
Pierce Fast Blocking Buffer	Single purified protein	Blocks in 5 minutes	When time is essential	TBS
SuperSignal Western Blot Enhancer	Membrane treatment for low abundance or poor immunoreactivity antibodies	Pre-treatment for nitrocellulose membranes Reduces the amount of primary antibody required for probing	Use when primary antibody is 3 to 100-fold less primary antibody than is usually used to detect the protein of interest	Ready-to-use

 

Stripping and re-probing of Western blots offers several advantages:

1. Conservation of samples that are expensive or available only in limited quantities;
2. Analysis of a given blot using several different antibodies, e.g. subtype- or isoform-specific antibodies;
3. Re-analysis of anomalous results and confirmation with the same or a different antibody;
4. Correcting errors in incubation with the wrong antibody;
5. Cost savings in reagents and time by reusing the same blot.

Neither of these methods will remove the colored precipitate resulting from chromogenic detection systems (eg, BCIP, 4CN, DAB and TMB). However, it is still possible to analyze the blot with another antibody specific for a different target protein.

Typically, these protocols should be used for qualitative purposes only, until it is established that stripping does not quantitatively affect a given antigen. Depending on the method and type of membrane used, many antigens will withstand at least 5 stripping cycles. However, it should be kept in mind that small portions of membrane-stabilized proteins will be removed during each stripping cycle. When multiple antigens are detected sequentially, it is recommended to start with antigens that are expected to be in low abundance or to give a low signal.

Here are some additional recommendations when planning a Western blot experiment with one or more rounds of antibody stripping.

• PVDF membranes are more robust than nitrocellulose and are therefore recommended for any protocol involving antibody stripping.
• Drying of PVDF membranes immediately after transfer from the SDS-PAGE gel improves binding of proteins to the membrane and is particularly recommended when multiple stripping is planned. Dry PVDF membranes must be rewetted with alcohol prior to the first round of immunodetection.
• Detect low-abundance antigens first.
• Use low-affinity antibodies before high-affinity antibodies.
• Important: Although drying of a PVDF blot is recommended immediately after transfer, the blot should not be allowed to dry between rounds of immunodetection. Any residual antibody molecules will bind permanently to the membrane if it is allowed to dry.

 

WB / Antibody Stripping Buffer
abx090671-200ml	Abbexa	200 ml	EUR 189

West Ez Stripping Buffer
S2100-006	GenDepot	60ml	EUR 81

West Ez Stripping Buffer, Economic Size
S2100-050	GenDepot	500ml	EUR 212

West Ez Stripping Buffer, Economic Size
S2100-100	GenDepot	2x500ml	EUR 280

Western blot recycling stripping buffer, 50 ml (10X)
90101	Alpha Diagnostics	50 ml	EUR 238

Phospho Antibody Stripping Solution
AKR-102	Cell Biolabs	1 kit	EUR 392
Description: This reagent allows you to strip phospho antibodies from protein blots and subsequently re-probe the same blot.

101Bio WB Stripping Solution
P5W3	101Bio	-	Ask for price

T-Pro Western Blot Stripping Reagent
JB11-K002	T-Pro Biotechnology	500ml/BT	EUR 178

Antibody diluent buffer
AR1106-2	BosterBio	12ml	EUR 91

Fixation Buffer
22015	Biotium	100mL	EUR 149
Description: Minimum order quantity: 1 unit of 100mL

Permeabilization Buffer
22016	Biotium	100mL	EUR 149
Description: Minimum order quantity: 1 unit of 100mL

RIPA Buffer
2114-100	Biovision		EUR 158

RIPA Buffer
2114-500	Biovision		EUR 463

Dilution Buffer
1066-100	Biovision		EUR 153

Dilution Buffer
1066-400	Biovision		EUR 262

Wash Buffer
1210-200	Biovision		EUR 196

Wash Buffer
abx098952-20ml	Abbexa	20 ml	EUR 91

Coating Buffer
abx098970-1vial	Abbexa	1 vial	EUR 154

Blocking Buffer
abx098972-1vial	Abbexa	1 vial	EUR 154

Lysis Buffer
abx098984-LysisBuffer120ml	Abbexa	Lysis Buffer 1 (20 ml)	EUR 154

Lysis Buffer
abx098984-LysisBuffer3100ml	Abbexa	Lysis Buffer 3 (100ml)	EUR 230

Lysis Buffer
abx098984-LysisBuffer420ml	Abbexa	Lysis Buffer 4 (20 ml)	EUR 154

Tricine buffer
20-abx082551	Abbexa	EUR 217.00 EUR 189.00	100 g 25 g

Binding Buffer
BB10X-50ML	ImmunoStep	50 ML	EUR 113.7