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

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.

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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.

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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.

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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.

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

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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.

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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.

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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).

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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.

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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

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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.

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