Invitrogen™ Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 680

Catalog No.	Quantity
A21076	1 mg

Catalog No. A21076 Supplier Invitrogen™ Supplier No. A21076

Description

Goat Polyclonal Secondary Antibody

To minimize cross-reactivity, these goat anti-rabbit IgG (H+L) whole secondary antibodies have been affinity purified and cross-adsorbed against human IgG, human serum, mouse IgG, mouse serum, and bovine serum. Cross-adsorption or pre-adsorption is a purification step to increase specificity of the antibody resulting in higher sensitivity and less background staining. The secondary antibody solution is passed through a column matrix containing immobilized serum proteins from potentially cross-reactive species. Only the nonspecific-binding secondary antibodies are captured in the column, and the highly specific secondaries flow through. The benefits of this extra step are apparent in multiplexing/multicolor-staining experiments (e.g., flow cytometry) where there is potential cross-reactivity with other primary antibodies or in tissue/cell fluorescent staining experiments where there are may be the presence of endogenous immunoglobulins. Alexa Fluor dyes are among the most trusted fluorescent dyes available today. Invitrogen™ Alexa Fluor 680 dye is a bright, near-infrared-fluorescent dye with excitation ideally suited to the 680 nm laser line. For stable signal generation in imaging and flow cytometry, Alexa Fluor 680 dye is pH-insensitive over a wide molar range. Probes with high fluorescence quantum yield and high photostability allow detection of low-abundance biological structures with great sensitivity. Alexa Fluor 680 dye molecules can be attached to proteins at...

Anti-Rabbit secondary antibodies are affinity-purified antibodies with well-characterized specificity for rabbit immunoglobulins and are useful in the detection, sorting or purification of its specified target. Secondary antibodies offer increased versatility enabling users to use many detection systems (e.g. HRP, AP, fluorescence). They can also provide greater sensitivity through signal amplification as multiple secondary antibodies can bind to a single primary antibody. Most commonly, secondary antibodies are generated by immunizing the host animal with a pooled population of immunoglobulins from the target species and can be further purified and modified (i.e. immunoaffinity chromatography, antibody fragmentation, label conjugation, etc.) to generate highly specific reagents.

Specifications

• Antigen: Rabbit IgG (H+L) Cross-Adsorbed
• Applications: Western Blot, Immunocytochemistry
• Classification: Polyclonal
• Concentration: 2 mg/mL
• Conjugate: Alexa Fluor 680
• Formulation: PBS with 5mM sodium azide; pH 7.5
• Host Species: Goat
• Immunogen: Gamma Immunoglobins Heavy and Light chains.
• Purification Method: Purified
• Quantity: 1 mg
• Regulatory Status: RUO
• Primary or Secondary: Secondary
• Target Species: Rabbit
• Content And Storage: 4°C, store in dark
• Product Type: Secondary Antibody
• Form: Liquid
• Isotype: IgG

Frequently Asked Questions (FAQs)

I am getting non-specific binding with my Alexa Fluor 790 and 680 reagent. Can you offer some tips?

Here are possible causes and solutions:

- Membrane contaminated by fingerprints or keratin proteins: Wear clean gloves at all times and use forceps when handling membranes. Always handle membranes around the edges.
- Concentrated secondary antibody used: Make sure the secondary antibody is diluted as recommended. If the background remains high, but with strong band intensity, decrease the concentration of the secondary antibody.
- Concentrated Primary antibody used: Decrease the concentration of the primary antibody.
- Affinity of the primary antibody for the protein standards: Check with the protein standard manufacturer for homologies with primary antibody.

How can I improve the signal intensity when using Alexa Fluor 790 and 680 detection reagents?

Here are possible causes and solutions for weak/no signal:

- Poor or incomplete transfer: Check transfer conditions, and repeat blot. Use positive control and/or molecular weight marker.
- Nitrocellulose membrane not completely wetted, or PVDF membrane not completely reactivated: Follow instructions for pre-wetting or reactivating the membrane.
: Secondary antibody concentration too low: Use the recommended secondary antibody concentrations.
- Primary antibody concentration too low: Use twice the concentration of primary antibody required for a standard immunodetection. If the signal is still low and the background is not high, increase the concentration.
- Inactive primary antibody: Determine activity by performing a dot-blot or other methods.
- Low affinity of primary antibody to antigen: Obtain a higher affinity primary antibody.
- Sample improperly prepared; antigenicity weakened, or destroyed: SDS and reducing agents may interfere with some antibody/antigen affinities.
- Sample too dilute: Load a higher concentration or amount of protein onto the gel.
- Blots are too old: Protein may have broken down over time. Use freshly prepared blots.
- Protein of interest ran off the gel: Match gel separation range to the size of the protein being transferred.
- Poor retention of proteins: Larger proteins require more transfer time, while smaller proteins require less transfer time. Use a molecular weight marker with relevant size proteins. Use membrane with the appropriate binding capacity.
- Alexa Fluor 790 and 680 reagents have been repeatedly frozen: Repeated freeze/thawing can cause antibodies to irreversibly precipitate. For long-term storage, it is best to aliquot into individual use tubes before freezing.

I am getting high background after Alexa Fluor 790 and 680 detection. Can you please offer some tips?

Here are possible causes and solutions:

- Insufficient blocking or non-specific binding: Try a different blocking reagent or increase the concentration of the blocking reagent. We generally obtain good results with 2% casein, 5% non-fat dry milk, or 1/2X fish serum.
- Membrane was blocked with BSA: Do not use BSA-containing solutions for blocking or incubating Alexa Fluor 680 and 790 conjugates. For primary antibodies that are incompatible with casein or milk (e.g., many anti-phosphoprotein antibodies), use fish serum or use a 0.5% BSA-containing solution for primary antibody incubation only and then switch to 2% casein or 5% non-fat milk for all other incubation steps.
- Membrane is contaminated: Use only clean, new membranes. Wear clean gloves at all times and use forceps when handling membranes.
- Higher intrinsic background with PVDF membranes: Switch to nitrocellulose membranes.
- Nitrocellulose membrane not completely wetted: Follow instructions for pre-wetting the membrane.
- Insufficient washing: Follow recommended number of washes. In some cases, it may be necessary to increase the number or duration of washes.
- Concentration of primary and/or secondary antibody is too high: Determine optimal antibody concentration by performing a dot blot and dilute antibody as necessary.

I am getting high background with Alexa Fluor 790 and 680 secondary conjugates. What can I do to reduce background?

Add 0.1% SDS to blocker for secondary antibody incubation step to further reduce nonspecific background staining. Use lower fluorescent PVDF-FL membranes rather than PVDF.

Can I image my Alexa Fluor 680 and 790-stained blots while they are wet?

Yes, Alexa Fluor 680 and 790-stained blots can be imaged wet or dried. We recommend drying blots for long-term storage.

How many western blots can I stain with the Alexa Fluor 680 and 790 secondary conjugates?

All Alexa Fluor 680 and 790 secondary conjugates come supplied as 0.5 mL of a 2 mg/mL stock or as a 1 mg powder, which is sufficient to stain 200 blots at a working concentration of 0.5 µg/mL and 10 mL/blot.

What working concentration of Alexa Fluor 680 and 790 secondary antibody or streptavidin conjugate should I use for western detection?

A good initial working concentration is ~0.5 µg/mL, which is a 1:4000 dilution of the 2 mg/mL stock. Depending on the abundance of your target, the optimal concentration may be in the range of 0.1-1 µg/mL.

Can I use western blot processing instruments, like the iBind device or BenchPro 4100 Card Processing Station with Alexa Fluor 680 and 790 secondary antibodies?

Yes, western blot processing instruments work well with Alexa Fluor 680 and 790 labeled secondary antibodies and give similar or better sensitivity compared to manual processing. See Figure 2 in the following link (http://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/detect-proteins-western-blot/ibind-western-system.html?icid=fr-western-3).

Can I use other colors of Alexa Fluor labeled antibodies, other than far-red Alexa Fluor 680 and 790 antibodies to do western detection?

Technically, the non-far red Alexa Fluor antibodies could be used on western blots, but they will give very poor sensitivity compared to other detection methods and thus are not recommended. Blot membranes, especially PVDF, have a high fluorescent background, highest in the blue/green range, which increases noise and thus lowers sensitivity. Blocking proteins can also autofluoresce, increasing background. Blot fluorescent background is very low in the far-red range, which is why Alexa Fluor 680 and 790 dyes can obtain high sensitivity. A second reason why non-far red Alexa Fluor dyes do not make good western blot detection reagents is that the non-Alexa Fluor 680 and 790 antibody conjugates are optimized to give high signals for immunofluorescence (IF) and immunohistochemistry (IHC) staining and generally have a high degree of dye:antibody labeling, which can lead to high nonspecific charge-based binding of the dyes to western blotted proteins and the membrane, increasing background staining and thus lowering signal to noise. The Alexa Fluor 680 and 790 secondary antibody conjugates have degrees of labeling that are optimized for western detection, so they have a conjugation efficiency that gives a high signal with lower nonspecific binding.

How does western detection with Alexa Fluor 680 and 790 secondary antibodies compare to chemiluminescent detection?

Alexa Fluor 680 and 790 secondary antibody reagents have similar sensitivity as ECL chemiluminescent detection. The sensitivity is in the picogram range. Far-red fluorescent detection has a number of advantages over enzyme-based chemiluminescent or chromogenic detection methods:

- Ability to do multiplex detection on the same blot at the same time
- Simple protocol that is not time-sensitive and does not require mixing of reagents
- Reliable protocol that can never give over- or under-developed blots
- High photostability enabling dried blots to be archived
- Uses more commonly available UV or blue light imagers rather than more-expensive chemiluminescent imagers or film and chemicals
- Sharper bands due to the direct linking of the far-red secondary antibody conjugate to the primary antibody target protein, rather than the diffuse edges around a protein band seen with enzyme-based detection methods

How does far-red western detection with Alexa Fluor 680 and 790 secondary antibodies compare to the Li-COR IRDye 680 and 800 dyes?

Detection sensitivity with Alexa Fluor 680 and 790 conjugated secondary antibodies is comparable to detection with Li-COR IRDye 680 and 800 dyes used at the same concentration.