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Enzyme-conjugated primary antibodies may not bind efficiently with the proteins in the MagicMark XP Western Protein Standard. We recommend using unconjugated primary antibody, followed by the addition of enzyme-conjugated secondary antibody.+
Note: The Anti-myc-AP/HRP and Anti-V5-AP/HRP antibodies do not bind to MagicMark XP proteins.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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Here are some suggestions:

- Verify that the detection reagents are working well. Optimize the antibody concentration to obtain best results.
- Make sure that the amount of standard loaded on the gel is correct.
- Optimize the transfer conditions (current, voltage, transfer time).
- Enzyme-conjugated primary antibodies may not bind efficiently with the proteins in the MagicMark XP Western Protein Standard. We recommend using unconjugated primary antibody, followed by the addition of enzyme-conjugated secondary antibody.
Note: The Anti-myc-AP/HRP and Anti-V5-AP/HRP antibodies do not bind to MagicMark XP proteins.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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Secondary antibodies may be either too specific (e.g., recognize only one host species of primary antibody) or too general (e.g., recognize whole IgG and any fragments thereof). In most cases, these limitations can be overcome by carefully designing the experimental system and choosing the appropriate secondary antibody. The following considerations are useful to help choose a secondary antibody:

Determine the host species of the primary antibody (e.g., mouse anti-tubulin).
Select an appropriate host species for the secondary antibody-you will need a secondary antibody that is raised in a species different from the host species of the primary antibody (e.g., goat anti-mouse IgG).
Consider cross-reactivity or specificity issues of the secondary antibody.
Highly cross-absorbed-for multiple-labeling applications or when using samples with endogenous antibodies.
Specificity-binds to correct fragments, classes, or chains of the primary antibody.
Use an appropriate detection or purification method.
Label-appropriately conjugated to the correct enzyme, tag, or fluorophore for the chosen detection method.
Ability to bind to Protein A, Protein G, or Protein L-make sure the secondary antibody chosen has sufficient affinity for the molecules used upstream or downstream (i.e., Protein A-coated microplates).
Consider requirements of the supplied secondary antibody.
Supplied state-sterile liquid or lyophilized, suspended in PBS or Tris buffer, contains carrier proteins such as gelatin or albumin or the addition of stabilizers such as sucrose or microbial inhibitors.

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A blocking step should be performed to reduce fluorescence due to non-specific antibody binding. A common blocking step is the addition of a 2-5% solution of bovine serum albumin (fraction V defatted BSA). Another approach employs the addition of a 5-10% solution of serum from the species in which the secondary antibodies were raised. For example, when using goat anti-mouse IgG secondary antibodies, samples may be effectively blocked with 5-10% normal goat serum. To further reduce background fluorescence, the Image-iT FX Signal Enhancer can be included as a pre-blocking step to decrease non-specific labeling due to charge interactions between the dyes on the conjugates and the cellular constituents.
If you are using a secondary antibody make sure that the species of the antibody is not the same as the species of the sample. For example do not use an anti-mouse secondary antibody on mouse tissue.
Titrate the antibody to the lowest concentration you can use and still get adequate signal.
Try using a fluorescently tagged primary antibody because it should give reduced background but be aware this can reduce signal intensity.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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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 WesternDot 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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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Both techniques rely on the efficiency and specificity of nucleic acid hybridization to locate specific DNA and RNA sequences in cells and tissues. FISH, or fluorescence in situ hybridization, relies on DNA or RNA probes directly labeled with fluorophores or with biotin. When biotin labels are used, detection is achieved with fluorophore-labeled biotin binding proteins like streptavidin. Imaging in FISH requires an epifluorescence microscope equipped with the appropriate filters for visualizing the light emitted by the fluorophores used. Typically, fluorescence microscopes need to be equipped with a CCD camera and the results are usually processed with image analysis software. One distinct advantage of FISH is that multicolor staining (multiplexing) is possible.

CISH relies on DNA and RNA probes labeled with non-fluorescent haptens like biotin and digoxigenin. These haptenylated probes are detected in a second step using streptavidin or anti-hapten antibodies conjugated to enzymes like HRP and alkaline phosphatase. Chromogenic substrates for these enzymes are used for localizing the target nucleic acids in the sections. Such chromogens as DAB, AEC, NBT/BCIP, and Fast Red yield insoluble, colored precipitates which stain the nucleic acid targets. 

An ordinary light microscope can be used to image CISH results, and unlike FISH, histological evaluation of the tissues is possible simultaneously if the slides are counterstained appropriately. Although multicolor CISH has been performed, it is much more difficult to do successfully, compared to FISH. However, unlike FISH, slides stained with CISH can be archived like any other pathology slides.

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