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Invitrogen™ WesternBreeze™ Chromogenic Kit, anti-rabbit

Catalog No. WB7105
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WB7105 1 kit
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Catalog No. WB7105 Supplier Invitrogen™ Supplier No. WB7105
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Description

Detect proteins that have been immobilized on membranes (nitrocellulose or PVDF) following western transfer or bound directly from solution (dot blots)

Detection is accomplished with a ready-to-use BCIP/NBT substrate for alkaline phosphatase. Non-fading purple precipitates develop at the protein bands on the membrane.

WesternBreeze™ chromogenic offers:

  • Clear background
  • High sensitivity-low picogram levels detectable
  • High specificity
    One simple protocol-no optimization required

Proteins, Expression, Isolation and Analysis, Western Blotting

Specifications

Product Line WesternBreeze
Content And Storage The WesternBreeze Chromogenic kits include blocking solutions, primary antibody diluent, ready-to-use secondary antibody solution (anti-mouse, anti-rabbit or anti-goat), ready-to-use chromogenic substrate, wash solutions, and incubation trays. Each kit contains complete reagents for 20 mini-blots.

Store the kits at 2–8°C. Guaranteed stable for 6 months when properly stored.
Membrane Compatibility Nitrocellulose, PVDF
Product Type Western Blot Kit
Detection Method Chromogenic
Substrate Type AP (Alkaline Phosphatase) Substrate
Shipping Condition Wet Ice
Reactivity Rabbit
Cross Reactivity Rabbit
Quantity 1 kit

Frequently Asked Questions (FAQs)

Why is the actual band size on a western blot different from the predicted size of the protein?

Western blotting is based on the separation of proteins by their size on a gel. However, migration of proteins through the gel matrix is also affected by other factors, which may cause the observed band size to be different from the predicted size.

Common causes are:
-Post-translational modification; for example phosphorylation and glycosylation increase the size of the protein
-Post-translation cleavage; many proteins are synthesized as precursor proteins, and then cleaved to give the active form
-Multimers, for example dimerization of a protein. This is usually prevented under reducing conditions, although strong interactions can result in the appearance of higher bands
-Splice variants; alternative splicing may result in different sized proteins being produced from the same gene
-Relative charge; the composition of amino acids (charged vs. non-charged)
What are the standard lysis buffers used with mammalian cells for detection of protein expression by immunoprecipitation (IP) or Western blot analysis?

The most commonly used buffer is RIPA Buffer with SDS. We offer RIPA Buffer (Cat. Nos. 89900 and 89901). We also offer the Pierce IP Lysis buffer (Cat. Nos. 87787 and 87788) as well as M-PER (Cat. Nos. 78501, 78503, and 78505).
What conditions do you recommend for overnight Western transfer?

Doing an overnight Westerm transfer is not the preferred method but can be done. The power should be lowered and the buffer should be chilled or the unit should be placed in the cold room to prevent overheating. You may try an overnight transfer at 5-15 V and adjust accordingly. You may also wish to put a second membrane behind the first in order to bind any proteins that transfer through the first membrane. You can use both membranes for staining, immunoblotting, or analysis.
Can I purchase the Secondary Antibody solutions from the WesternBreeze Chromogenic Detection kits as standalone products?

The goat anti-mouse and goat anti-rabbit secondary antibody solutions AP-conjugated are available as standalone products (Cat. Nos. WP20006 and WP20007) but the rabbit anti-goat secondary antibody solution AP-conjugated is not available as a standalone product.
Can I purchase the Blocker/Diluent and Antibody Wash solutions from the WesternBreeze Chromogenic Detection kits as standalone products?

Yes, you may purchase them as standalone products using the Cat. Nos. listed below:

- Cat. No. WB7003 (Antibody Wash)
- Cat. No. WB7001 (Blocker/Diluent A)
- Cat. No. WB7002 (Blocker/Diluent B)
- Cat. No. WB7050 (Combo pack containing Blocker/Diluents A & B)
How should I prepare my membranes for WesternBreeze Chromogenic detection?

For western blots, where proteins are freshly transferred from SDS-PAGE gels to nitrocellulose or PVDF membranes, washing the membranes twice for 5 mins with 20 mL of pure water is recommended to partially remove gel and transfer buffer components and weakly bound proteins. The membranes are then ready for the WesternBreeze Chromogenic Immunodetection protocol.

Alternatively, the washed membranes may be dried on a clean piece of filter paper in open air, by a stream of slightly warm air or under an infrared lamp. Properly dried membranes can be stored in a closed container at 4 degrees C for several days depending on the antigen loaded. Water-washed and dried nitrocellulose membranes are ready for the WesternBreeze Chromogenic Immunodetection protocol. However, water-washed and dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 mins before proceeding to the WesternBreeze Chromogenic Immunodetection protocol.

For Native-PAGE western blot, a drying step, performed before any washing steps, is recommended to improve protein binding to the membrane. Once dried, nitrocellulose membranes should be washed twice with 20 mL water for 5 mins before proceeding to the WesternBreeze Chromogenic Immunodetection protocol. Dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 minutes before proceeding to the WesternBreeze Chromogenic Immunodetection protocol.
Is the Chromogenic Substrate from the WesternBreeze Chromogenic Detection Kits (BCIP/NBT) available as a standalone product?

Yes, you can purchase it as a standalone (Cat. No. WP20001). It will give a purple chromogenic precipitate.
What are the different types of WesternBreeze Chromogenic Detection kits that you offer?

We offer the following three types of WesternBreeze Chromogenic Detection Kits:

- WesternBreeze Chromogenic Detection Kit: Anti-Mouse, Cat. No. WB7103
- WesternBreeze Chromogenic Detection Kit: Anti-Rabbit, Cat. No. WB7105
- WesternBreeze Chromogenic Detection Kit: Anti-Goat, Cat. No. WB7107

For Research Use Only. Not for use in diagnostic procedures.