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A western blot kit is not one standard product. Depending on the catalog number, it can be a set of detection reagents for a membrane you have already blotted, or a matched set of detection stacks and reagents built around a specific instrument. Both kinds are sold for research use only. This page sorts the kit types by what they cover, lists what the manufacturer manuals say is in the box and what is not, and notes the handling points the manuals themselves stress.
Who uses it: bench scientists, lab managers and core-facility staff who run immunodetection on blotted membranes, and buyers comparing catalog entries.
What a western blot kit is
In the Invitrogen WesternBreeze manual, the kit is described as containing complete, optimized, ready-to-use or ready-to-dilute reagents for sensitive immunodetection of western blots or dot blots, detecting primary antibodies on nitrocellulose or PVDF membranes. In that design the kit starts after protein transfer: you supply the blotted membrane and the primary antibody. The Thermo Fisher iBlot Western Detection Kit manual is likewise labelled “For research use only. Not intended for human or animal diagnostic or therapeutic uses.”
For the surrounding workflow (gel, transfer, blocking, antibodies), see western blot protocol basics. This page covers only the packaged-kit question.
What is usually inside
The Invitrogen WesternBreeze and iBlot manuals show the same building blocks in different forms: buffers for blocking and antibody dilution, a wash solution, a labelled secondary antibody or antibody matrix, and a detection substrate. The substrate defines the detection chemistry. The WesternBreeze kit is chromogenic and uses BCIP/NBT as the substrate for an alkaline-phosphatase-conjugated secondary antibody. The iBlot manual covers both chemiluminescent and chromogenic kits.
WesternBreeze chromogenic kit (manual version F, 2003)
- Blocker/Diluent Parts A and B, concentrated buffered saline solution containing detergent
- Antibody Wash (16X), a concentrated Hammersten casein solution
- Chromogenic Substrate (BCIP/NBT for alkaline phosphatase), ready to use
- Secondary Antibody Solution, an alkaline-phosphatase-conjugated affinity-purified anti-species IgG (goat conjugate for the anti-mouse and anti-rabbit kits, rabbit conjugate for the anti-goat kit)
- Two plastic incubation dishes
Reagents supplied are enough to detect 20 mini-blots.
iBlot Western Detection Kit
The kit ships as two boxes. The Stack Box holds consumables for the stack, and the Reagent Box holds the solutions: wash solution (16X), antibody diluent, diluent additive and the substrate (a chemiluminescent substrate with enhancer, or a chromogen).
Kit types and variants
| Type | Example in the manual | What it covers | Key difference |
|---|---|---|---|
| Reagent-only chromogenic detection kit | Invitrogen WesternBreeze (cat. WB7103, WB7105, WB7107) | Blocking, wash, secondary antibody and BCIP/NBT substrate for NC or PVDF membranes; 20 mini-blots | No instrument tie; user supplies the blotted membrane, primary antibody and an orbital shaker |
| Matched stacks plus reagents | iBlot Western Detection Kit, chemiluminescent or chromogenic, anti-mouse or anti-rabbit, Regular or Mini, 10 Pak | Detection stacks with antibody matrix and the reagent box | Needs the iBlot device (firmware 2.9.5 or higher, program P9) |
| Stacks alone | iBlot Western Detection Stacks, Regular or Mini, 10 Pak | Stack Box items only | Reagents bought separately; detection only |
Within each family, the manuals separate variants by the species of the primary antibody the secondary recognizes (the WesternBreeze mouse, rabbit and goat kits; the iBlot anti-mouse and anti-rabbit kits). See secondary antibodies for western blot for how that choice works.
What is not in the box
This is the part catalog listings rarely spell out. The WesternBreeze manual lists as needed but not supplied: blotted membranes, primary antibody, purified water that is autoclaved, sterile or ultrafiltered to remove alkaline phosphatase activity, clean flasks, forceps and an orbital shaker platform. The iBlot manual states that the detection stacks are not for transfer: “Do not use Western Detection Stacks for protein transfer.” Protein must already be on a PVDF or nitrocellulose membrane. Gel electrophoresis, sample markers (see protein markers) and transfer are separate products.
Capacity, sizes and storage
| Item | Value stated in the manual |
|---|---|
| WesternBreeze capacity | Reagents for 20 mini-blots; some reagents are normally left over after 20 mini-blots because PVDF and nitrocellulose use different amounts |
| WesternBreeze storage | Solutions stored at 2-8 C; once diluted, use the same day |
| iBlot Stack Box capacity | Stacks for 10 mini blots (8 cm x 8 cm) or 10 regular blots (13.5 cm x 8 cm) |
| iBlot Stack Box contents | Stack Bottom 10, Stack Top 10, Disposable Sponge 10, Antibody Matrix 30, Transparent Sheet 40 |
| iBlot membrane size limit | Not larger than 8 cm x 8 cm (mini stacks) or 8 cm x 13 cm (regular stacks) |
| iBlot shipping and storage | Stack Box shipped and stored at room temperature; Reagent Box shipped on blue ice and stored at 4 C |
Sources: Invitrogen manual IM-1004 version F (June 2003) and the Invitrogen iBlot Western Detection Kits manual, both checked 2026-10-29. Current catalog numbers may carry a newer manual, so check the one for the exact product.
How to compare kits
Using only criteria the manuals state:
- Membrane type: the WesternBreeze kit is for nitrocellulose or PVDF membranes; the iBlot detection kits likewise assume protein already transferred to PVDF or nitrocellulose.
- Primary antibody species: kits are sold per species (mouse, rabbit, goat for WesternBreeze; mouse or rabbit for iBlot).
- Detection chemistry: chromogenic or chemiluminescent. Imaging implications are covered in film vs CCD vs laser scanner imaging.
- Blot size and capacity: stated in blots of a given size (mini 8 cm x 8 cm, regular 13.5 cm x 8 cm).
- Instrument requirement: the iBlot kits require the iBlot device with specified firmware; WesternBreeze does not name an instrument.
For reference, the WesternBreeze manual reports detection of 10-100 pg of human IgG on nitrocellulose after a 4-12% NuPAGE Bis-Tris gel; within 40 minutes of colour development it detected 10 pg with the rabbit kit and 100 pg with the mouse and goat kits. These are the manufacturer’s qualification figures for that kit, not a comparison across kits.
Safe use and handling basics
- The WesternBreeze manual notes the BCIP/NBT solution contains NBT, “a possible carcinogen”, and recommends gloves, safety glasses and a lab coat.
- The kit solutions contain a chlorobutanol preservative per the manual; follow the product’s safety data sheet.
- Store as stated: 2-8 C for WesternBreeze solutions, room temperature for the iBlot Stack Box and 4 C for its Reagent Box.
Common mistakes
- Reusing dishes. The manual says to use a single clean dish for each blot.
- Touching the membrane’s working surface, even with gloves.
- Letting the membrane dry. Membranes dry quickly; the manual says to re-wet a dried membrane with methanol.
- Using ordinary water with an alkaline-phosphatase system, when the manual calls for water treated to remove alkaline phosphatase activity.
- Expecting the kit to do transfer. The iBlot stacks are detection-only.
- Keeping diluted solutions overnight. The manual says to use them the same day.
If a run gives no signal, see the no-bands troubleshooting decision tree, and for quantification see loading control normalization.
FAQ
What does a western blot kit include?
It depends on the kit. A reagent-only kit such as WesternBreeze includes blocking and wash buffers, an enzyme-conjugated secondary antibody, a substrate and incubation dishes. Matched kits such as the iBlot detection kit add detection stacks and an antibody matrix.
Does a western blot kit include the gel, membrane or primary antibody?
In these two manufacturer manuals, no. The WesternBreeze manual lists blotted membranes and primary antibody as user-supplied, and the iBlot stacks are for detection only.
How many blots does one kit cover?
WesternBreeze supplies reagents for 20 mini-blots; the iBlot Stack Box covers 10 mini or 10 regular blots. Capacity is catalog-specific.
Are these kits for diagnostic use?
No. The iBlot manual states they are for research use only.
Sources
- Invitrogen, WesternBreeze Chromogenic Western Blot Immunodetection Kit instruction manual IM-1004, version F, June 2003 (lab-hosted copy: manual PDF), checked 2026-10-29.
- Invitrogen / Thermo Fisher Scientific, iBlot Western Detection Kits user manual (manual PDF), checked 2026-10-29.
Related reading: dot blot.
