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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:

- 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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If you are using more than one primary antibody, they need to be from different species, such as mouse and rabbit. If they are from the same species, your secondary antibody will label both primary antibodies. Your secondary antibodies need to be raised in a species other than the ones your primary antibodies are raised in. For example, if you have rabbit anti-target and mouse anti-target primary antibodies, you cannot use a rabbit anti-mouse secondary. This is because when you come in with your anti-rabbit secondary, it will label the rabbit anti-mouse secondary as well. There is no problem with using the same host for all of your secondary antibodies. In this example, use of goat anti-mouse and goat anti-rabbit secondary antibodies would be a good choice.

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Yes. Primary antibodies against two or three different antigens can be applied together (in a mixture) or sequentially.
- If using secondary antibodies for labeling, each primary antibody must be raised in a different species; if the primary antibodies are of the same species, they must be of different isotypes and one must use secondary antibodies that are selective for these isotypes.
- Each secondary antibody should be labeled with fluorophores that exhibit little or no overlap in their spectra.
- All of the secondary antibodies may be raised in the same species, such as using Donkey anti-mouse, Donkey anti-goat, and Donkey anti-rabbit antibodies to label primary antibodies raised in mouse, goat, and rabbit. Avoid using secondary antibodies of different species that may be labeled by the other secondary antibodies in the mixture. For example, when using three primary antibodies raised in mouse, rabbit, and goat, one cannot use secondary antibodies such as Goat anti-rabbit, Donkey anti-goat, and Rabbit anti-mouse antibody. These secondary antibodies will label their respective primary antibodies and each other. - Use highly cross-adsorbed secondary antibodies to avoid species cross-reactivity. Cross-reactivity is highest between these species: Horse-Donkey, Sheep-Goat and Rat-Mouse.

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In general, no signal or weak signal can be a result of one or more of the following:

(1) Reagents were omitted or added in an incorrect order. Use all reagents in the proper sequence.

(2) Incorrect reagents were used. Use matched reagents (for example, a mouse primary antibody with an anti-mouse secondary antibody).

(3) Insufficient amounts of antigen were present, which prevented detection with the immunodetection procedure employed. You could:
-Use a more sensitive detection system.
-Use a secondary antibody instead of a directly labeled primary antibody. This may increase the signal 10-fold or more.
-Increase the primary antibody concentration.
-Increase the incubation time of the primary antibody with the antigen.
-Use more antigen.

(4) Improper storage of reagents resulted in degradation. Store reagents at recommended conditions.

(5) Low-affinity primary antibody was lost during immunodetection procedure. Try higher affinity antibody if available. Increase the incubation time or concentration of primary antibody with the antigen to maximize the amount of primary antibody bound. Decrease wash volumes and/or times to decrease dissociation of the primary antibody.

(6) Primary antibody reacted poorly with denatured antigen. Use procedure and buffers that allow retention of the native form of the antigen.

(7) Incubation times with secondary antibody or the streptavidin or avidin conjugate were insufficient. Increase these incubation times.

(8) Incorrect incubation temperatures were used in one or more of the steps. Use recommended incubation temperatures.

(9) Horseradish peroxidase conjugate activity was inhibited. Do not use sodium azide in the conjugate dilution buffer and the substrate buffer. Use the recommended concentration of hydrogen peroxide. The resulting signal will be reduced if either insufficient or excessive amounts are employed.

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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.
- Insufficient removal of SDS or weakly bound proteins from membrane after blotting: Follow instructions for membrane preparation before immunodetection.
- Short blocking time or long washing time: Make sure that each step is performed for the specified amount of time.

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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.
- Primary antibody too concentrated: Follow the supplier's recommended dilution or determine the optimum concentration by dot blotting.
- Insufficient removal of SDS/weakly bound proteins from membrane after blotting: Follow instructions for membrane preparation before immunodetection, as directed in the manual.
- Affinity of the primary antibody for the protein standards: Check with protein standard manufacturer for homologies with primary antibody.
- Improper preparation of iBind Solution/ iBind FD Solution: Prepare 1X iBind Solution/ iBind FD Solution as directed in the manual.

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Yes, a cocktail of primary antibodies can be added to a cell suspension in order to pull out several target cell populations with one secondary-coated Dynabeads magnetic beads product.
The Dynabeads magnetic beads Pan Mouse IgG (110.41; 110.42) works very well with a cocktail of mouse IgGs for the simultaneous capture of multiple cell types. it is recommended that you use an indirect technique with antibody cocktails (add all Ab to cells, wash off excess Ab, then add beads to capture Ab-coated cells).

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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.
- Primary antibody too concentrated: Follow the supplier's recommended dilution or determine the optimum concentration by dot blotting.
- Insufficient removal of SDS/weakly bound proteins from membrane after blotting: Follow instructions for membrane preparation before immunodetection, as directed in the manual (https://tools.thermofisher.com/content/sfs/manuals/ibind_flex_man.pdf).
- Affinity of the primary antibody for the protein standards: Check with protein standard manufacturer for homologies with primary antibody.
- Improper preparation of iBind Flex Solution/ iBind Flex FD Solution: Prepare 1X iBind Flex Solution/ iBind Flex FD Solution as directed in the manual.

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Our secondary antibody-coated Dynabeads magnetic beads are pre-coated with affinity purified polyclonal secondary antibodies. We offer the following secondary antibody-coated Dynabeads magnetic beads:

-Dynabeads M-280 Sheep anti-Rabbit IgG (Cat. No. 11204D): 2.8 µm beads to be used when your primary antibody is derived from rabbits
-Dynabeads M280 Sheep Anti-Mouse IgG (Cat. No. 11201D): 2.8 µm beads to be used when your primary antibody is derived from mice
-Dynabeads Sheep anti-Rat IgG (Cat. No. 11035): larger 4.5 µm beads to be used when your primary antibody is derived from rats; please note that smaller beads provide a larger surface area and hence would give higher yields of protein
-Dynabeads Pan Mouse IgG (Cat. No. 11041): 2.8 µm beads that will bind all subclasses of mouse IgG

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The secondary-coated Dynabeads magnetic beads can be coupled to a primary antibody by a direct or an indirect approach.
Direct approach: The Dynabeads magnetic beads are first coupled with your primary antibody and then used for isolating your target cell type.
Indirect approach: The cells are first incubated with your primary antibody(ies). The Dynabeads magnetic beads are then added to the antibody-coated target cells.

Secondary-coated Dynabeads magnetic beads can be used in several cell isolation approaches:
Cell depletion--using an antibody to target the unwanted cell type and a secondary-coated Dynabeads magnetic beads product.
Negative cell isolation--using a cocktail of antibodies to target all unwanted cell types and a secondary-coated Dynabeads magnetic beads product (using the indirect approach).
Positive cell isolation without detachment--using an antibody to target the wanted cell type and a secondary-coated Dynabeads magnetic beads product.
Positive cell isolation with detachment--using an antibody to target the wanted cell type and a CELLection Dynabeads magnetic beads product.

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Here are possible causes and solutions:

- Insufficient removal of SDS or weakly bound proteins from membrane after blotting: Follow instructions for membrane preparation before immunodetection.
- Short blocking time or long washing time: Make sure that each step is performed for the specified amount of time.
- Affinity of the primary antibody for the protein standards: Check with protein standard manufacturer for homologies with primary antibody.
- Membrane is contaminated by fingerprints or keratin proteins_ Wear clean gloves at all times and use forceps when handling membranes. Always handle membranes around the edges.

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No single buffer formulation works best for every protein/antibody:

- 2% casein, 5% non-fat dry milk, or 1/2X fish serum (e.g., SEA BLOCK) in 1x PBS/TBS work well for most target protein and antibody combinations.
- Do not use BSA-containing solutions for blocking or incubating WesternDot conjugates as their use may cause high background and/or reduced signal.
For primary antibodies that are incompatible with casein or milk (e.g., many antiphosphoprotein antibodies), use fish serum or a 0.5% BSA-containing solution.
If using BSA, use for the primary antibody incubation only; in all other steps, use 2% casein, 5% non-fat milk or 1/2x fish serum.

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We offer the following secondary antibody-coated Dynabeads magnetic beads:

-Dynabeads M-280 Sheep anti-Rabbit IgG (Cat. No. 11204D): 2.8 µm beads to be used when your primary antibody is derived from rabbits
-Dynabeads M280 Sheep Anti-Mouse IgG (Cat. No. 11201D): 2.8 µm beads to be used when your primary antibody is derived from mice
-Dynabeads Sheep anti-Rat IgG (Cat. No. 11035): 4.5 µm beads to be used when your primary antibody is derived from rats.
-Dynabeads Pan Mouse IgG (Cat. No. 11041): 4.5 µm beads that will bind all subclasses of mouse IgG

Although Dynabeads Sheep anti-Rat IgG and Dynabeads Pan Mouse IgG may be used for immunoprecipitation, please note that smaller (2.8 µm) beads provide a larger surface area and hence would give higher yields of protein than 4.5 µm beads.

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One thing you should do is to permeabilize well, by including 0.1% to 0.2% Triton X-100 in all washes and incubations. Another recommendation is to extend the labeling times to overnight at 4^oC for both primary antibody and secondary antibody. Finally, label the section free-floating, rather than adhered to a slide, to gain penetration for the antibody from both sides.

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