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There are two types of protocols for immunodetection: Standard and rapid.
|Step||Standard Immunodetection||Rapid Immunodetection|
|Block the membrane||1 hr||None|
|Incubate with primary antibody||1 hr||1 hr|
|Wash the membrane||3 x 10 min||3 x 5 min|
|Incubate with second antibody||1 hr||30 min|
|Wash the membrane||3 x 10 min||3 x 5 min|
|Add substrate||5 min||5 min|
|Total time||4 hr 5 min||2 hr 5 min|
Rapid immunodetection eliminates the blocking step and reduces the time necessary for the washing and incubation steps. The rapid immunodetection method works well to quickly visualize higher abundance proteins. Standard immunodetection, however, offers higher sensitivity and requires less optimization for new sample types.
Standard immunodetection is performed on blotted proteins directly after electrotransfer. (If the membrane was dried after transfer, thoroughly wet the blot for 1 minute in methanol if using PVDF or Milli-Q water if using nitrocellulose before proceeding to immunodetection.)
The unoccupied membrane binding sites on the wet blot are blocked with optimized reagents. The drawbacks of this method are the need for blocking and the total time requirement of over 4 hours. The advantage is that standard immunodetection may require less optimization for new sample types.
The following is a general protocol for fluorescent immunodetection. For optimal results, refer to manufacturer’s protocol provided with the reagents.
Rapid immunodetection takes advantage of the fact that antibodies cannot bind to the hydrophobic (non-wetted) surface of the Immobilon-P transfer membrane, but will bind to a protein immobilized on the membrane. Rapid immunodetection is compatible with both chromogenic and chemiluminescent substrates. The major advantage of rapid immunodetection is that blocking is not required, saving time and eliminating the risks involved (Mansfield, 1994). The method can be used on both dry and wet Immobilon PVDF membranes, enabling the analysis in about 2 hours, as opposed to over 4 hours for the standard method.
High salt buffer: PBS or TBS buffer supplemented with 0.5 M NaCl and 0.2% SDS
Two protocols for removing antibodies from blots are presented below. The first is applicable to any chemiluminescent substrate system and uses a combination of detergent and heat to release the antibodies. The second is commonly used for applications where antibodies have to be separated from an antigen and employs low pH to alter the structure of the antibody in such a way that the binding site is no longer active. Neither method will remove the colored precipitates generated from chromogenic detection systems (e.g., BCIP, 4CN, DAB and TMB). However, it is still possible to analyze the blot with another antibody specific to a different target protein.
The Re-Blot Plus Western Blot Recycling Kit contains specially formulated solutions that quickly and effectively remove antibodies from Western blots without significantly affecting the immobilized proteins.
The Re-Blot Plus Western Blot Recycling Kit (Cat. No.2500) is effective for removal of antibodies from Western blots that have been developed with chemiluminescence or radioactive iodine or other isotopes.
It is not recommended for stripping colorimetric substrates (TMB, DAB, 4-chloronapthol, etc.), as it is not possible to effectively remove substrates that precipitate at the reaction site. The Re-Blot Plus Western Blot Recycling Kit should be used only for qualitative purposes until it has been established by comparative blot analysis that stripping does not quantitatively affect a given antigen.
Kit components should be stored at 4°C upon arrival. Product is stable for 3 to 6 months after receipt. If Antibody Stripping Solution crystallizes upon storage, it may be re-dissolved with gentle warming at 37°C before use.
Re-Blot Plus Mild Stripping Solution gives good results on both nitrocellulose and PVDF membranes. However, Re-Blot Plus Strong Stripping Solution will perform better when membranes with high signal are to be stripped or when Re-Blot Plus Mild treatment is not sufficient.
|# of Strips or Blots||Amount of 10X Antibody Stripping Solution||Amount of Distilled Water||Resulting Amount of 1x Working Stripping Solution|
|1 Strip||400 μL||3.6 mL||4 mL|
|5 Strips||1 mL||9.0 mL||10 mL|
(7 x 10 cm)
|2 mL||18.0 mL||20 mL|
Immediately before use:
Dilute Antibody Stripping Solution (Mild or Strong) 10x with distilled water to obtain a 1x solution. If Antibody Stripping Solution contains crystals, warm gently at 37°C until crystals have dissolved completely. Prepare enough solution to allow free movement of strips or blots during incubation, typically 4 mL per strip or 20 mL per standard blot.
Use the following chart for suggested volumes of stripping or blocking solution.
Use this protocol when using antibodies used with competing immunogens.
Antibody gives good positive results at 2 μg/mL. Normally nitrocellulose is incubated in a final volume of 10 mLs. Combine 20 μg of the antibody with 100 μg of the peptide in 1mL of P(T)BS-milk. Incubate. Add the 1mL of peptide/antibody to 9mL of P(T)BS-milk.
* Preparing a 1mM stock of peptide to be further diluted:
PVDF is a chemically resistant polymer with excellent long term stability. For blots that need to be stored for use at a later date, storage conditions are determined by the stability of the proteins bound to the membrane. While Millipore recommends storage at low temperature, room temperature may be adequate for some proteins.
The azide must be thoroughly washed out of the blot prior to use as it inhibits HRP activity.
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