I used the iBind™ Flex Western system and am getting high background. What should I do?

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Answer

Here are possible causes and solutions:

- Membrane not completely wet: Follow instructions for prewetting the membrane. Use an incubation dish which is small enough to allow thorough coverage of membrane to prevent drying out. Note: If PVDF membrane is being used, we recommend making sure that it is activated with methanol, especially if it has dried up. It is not necessary to activate a PVDF membrane that has just come out of the transfer and moved into 1X iBind™ Flex Solution/ iBind™ Flex FD Solution.
- Membrane is contaminated: Use only clean, new membranes. Wear clean gloves at all times and use forceps when handling membranes.
- Film overexposed or became wet during exposure: Decrease exposure time or allow signal to further decay. Prevent leakage by encasing membrane in transparency film and blotting excess substrate from edges before exposure.
- Solutions or incubation tray are contaminated: Use clean glassware and purified water to prepare solutions. Replace or clean the tray thoroughly with a glassware-cleaning detergent. Rinse thoroughly with purified water. Wear clean gloves at all times.
- Concentrated primary antibody used: Follow the supplier's recommended dilution or determine the optimum concentration by dot blotting.
- Incorrect chemiluminescent substrate used for PVDF: Make sure CDP-Star™ reagent without enhancer is used.
- Blot is overdeveloped: Follow recommended developing time or remove blot from substrate when signal-to-noise ratio is acceptable.
- Ink used to label membrane: Any labeling of the membrane with ink should be limited to the low MW region of the blot.
- Improper preparation of iBind™ Flex Solution/ iBind™ Flex FD Solution: Prepare 1X iBind™ Flex Solution/ iBind™ Flex FD Solution as directed in the manual (https://tools.thermofisher.com/content/sfs/manuals/ibind_flex_man.pdf).
- Improper application of solutions to iBind™ Flex Wells: Add the appropriate solutions for each well in the correct numerical order as specified in the manual (https://tools.thermofisher.com/content/sfs/manuals/ibind_flex_man.pdf).
- Blot improperly placed on iBind™ Flex Card: 1) Place the membrane in the designated Membrane Region on the iBind™ Flex Card. 2) The protein side of the blot should be in contact with the iBind™ Flex Card. 3) The low MW regions should be closest to the Stack. 4) The membrane should not be in contact with the Stack.
- Card stack wet prior to run: Ensure that 10 mL of 1X iBind™ Flex/iBind™ Flex FD Solution is added to the flow region of the card. Avoid adding the solution to the stack.

Here are some additional tips to reduce background:

- If iBlot™ PVDF stacks are used, check that they have not expired as background increases with age. Using the iBlot™ 2 PVDF stacks will help in reducing the background.
- Make sure that the blot is rinsed in distilled water prior to adding the substrate. Do not rinse in PBS or TBS.

Answer Id: E11312

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With the iBind™ Flex Western System, I am getting a lot of non-specific binding. Can you offer some tips?

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Answer

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.

Answer Id: E11313

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Do you know where the HRP in anti-myc/HRP is conjugated?

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Answer

The conjugation was performed using glutaraldehyde crosslinking, which could potentially couple HRP anywhere on the antibody. However the conjugated antibody is tested to make sure there is no overall effect on antigen binding activity.

Answer Id: E3635

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If Thermo Fisher Scientific does not have a primary-coated Dynabeads™ magnetic beads product for isolating my target cell type, which alternative Dynabead™ magnetic beads product can I use instead?

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Answer

You may use one of our secondary-coated, surface-activated, or streptavidin-coated Dynabeads™ magnetic beads, and coat it with a primary antibody to target your cell type.

The Dynabeads™ magnetic beads product you choose will depend on the primary antibody available for cell targeting and the downstream application for the isolated cells:
-For primary antibodies made in mouse, use the CELLection™ magnetic beads Pan Mouse IgG Kit, Dynabeads™ magnetic beads Goat Anti-Mouse IgG, Dynabeads™ magnetic beads Pan Mouse IgG, Dynabeads™ magnetic beads Rat Anti-Mouse IgM, Dynabeads™ magnetic beads Rat Anti-Mouse IgM, or Dynabeads™ magnetic beads Sheep-Anti Mouse IgG

-For primary antibodies made in rat, use the Dynabeads™ magnetic beads Sheep Anti-Rat IgG

-For primary antibodies made in rabbit, use the Dynabeads™ magnetic beads M-280 Sheep Anti-Rabbit IgG

-For primary antibodies made in any species, use the CELLection™ magnetic beads Biotin Binder Kit, Dynabeads™ magnetic beads Biotin Binder, Dynabeads™ magnetic beads FlowComp™ Flexi, Dynabeads™ magnetic beads M-450 Epoxy, or Dynabeads™ magnetic beads M-450 Tosylactivated

Answer Id: E4670

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How long can for the CytoScan™ 750K Arrays be stored in holding buffer before scanning?

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Answer

After the washing and staining step, arrays can be stored for up to 24 hours at 40 degrees C before scanning.

Answer Id: E13923

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I used the iBind™ Flex Western System and am getting a very weak/almost no signal. What is the problem?

Product FAQ

Answer

Here are possible causes and solutions:

- Poor or incomplete transfer: Repeat blot. After blotting, stain membrane to measure transfer efficiency. Use positive control and/or molecular weight marker.
- Membrane not completely wet: Follow instructions for prewetting the membrane.
- Primary antibody concentration too low: Follow the supplier's recommended dilution or determine the optimum concentration by dot blotting.
- Inactive primary antibody: Determine activity by performing a dot-blot.
- Low affinity of primary antibody to antigen: Obtain a higher affinity primary antibody.
- Contaminated secondary antibody solution: Wear gloves at all times and keep bottles tightly capped when not in use. Use only purified water when preparing reagents.
- Protein of interest ran off the gel: Match gel separation range to size of protein being transferred.
- Poor retention of proteins: Match gel separation range to size of protein being transferred. Use a molecular weight marker with relevant size proteins. Larger proteins require more transfer time, smaller proteins less. Use membrane with the appropriate binding capacity.
- Improper preparation of iBind™ Flex Solution/ iBind™ Flex FD Solution: Prepare 1X iBind™ Flex Solution/ iBind™ Flex FD Solution as directed in the manual (https://tools.thermofisher.com/content/sfs/manuals/ibind_flex_man.pdf).
- Improper application of solutions to iBind™ Flex Wells: Ensure that the solutions are added to the correct wells and that the wells are loaded in numerical order.
- Blot improperly placed on iBind™ Flex Card: Ensure that the protein side of the blot is in contact with the iBind™ Flex Card and is placed in the region labeled “membrane”.
- Stack wet prior to run: Ensure that 5 mL of 1X iBind™ Flex Solution/ iBind™ Flex FD Solution is added to the flat region of the iBind™ Flex Card. Avoid adding solution to the Stack.
- Cross-contamination of solutions in wells: Do not move the iBind™ Flex Western Device during the run.
- iBind™ Flex Card damaged: Replace with new card. Ensure that rolling of the membrane on the card is limited to the area labeled “membrane”.
- Membrane is not in proper contact with the iBind™ Flex Card: Place the membrane on the iBind™ Flex Card immediately after adding a 1 mL pool of 1X iBind™ Flex Solution/ iBind™ Flex FD Solution. Use the roller provided to ensure proper contact.
- Device opened prior to completion of run: The device should not be opened once the card has been placed in the device. Re-sealing of the wells on the card can result in leaks.
- 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.
- Protein weakly bound to membrane: Ensure that transfer buffer contains 10-20% methanol.
- Insufficient exposure time: Re-expose film for a longer period of time.
- Insufficient substrate incubation: Perform each step for the specified amount of time or remove blot from substrate when signal-to-noise ratio is acceptable.
- Substrate is contaminated: Wear gloves at all times and keep bottles tightly capped when not in use.
- Blots are too old: Protein may have broken down over time. Use freshly prepared blots.

Answer Id: E11314

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Does the Anti-Thio™ antibody detect the His-Patch in the His-Patch ThioFusion™ System?

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Answer

Yes, the His-Patch vectors have been tested successfully with the Anti-Thio™ antibody in Western blot analysis.

Answer Id: E3636

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With regard to the promoter, H1 and U6, for the BLOCK-iT™ Lentiviral RNAi Expression System, is there a difference in knockdown?

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Answer

Constitutive knockdown is virtually identical for these two promoters in HEK 293 cells. In other cell types there are reports that either H1 or U6 may be more active, though in general, differences are minimal.

Answer Id: E10009

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How can I isolate cells using secondary-coated Dynabeads™ magnetic beads?

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Answer

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.

Answer Id: E4671

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What is the correct fluidics script for the CytoScan™ 750K Array?

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Answer

The correct fluidics script is the CytoScan™ 750K_Array_450.

Answer Id: E13924

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Do you have any reagents for canine studies?

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Answer

Thermo Fisher Scientific currently has over 150 products available for canine studies. There are 126 rabbit antibodies which recognize numerous canine antigens, among others; 50 of these are polyclonal while 76 are our recombinant monoclonal rabbit ABfinity™ antibodies. Fifty-three mouse monoclonal antibodies which recognize canine proteins, among others, are also available.

For customers who want to evaluate a panel of antibodies which bind to canine antigens, we have 2 popular antibody sampler packs. Catalog number 90-0900 contains a selection of 6 anti-claudin antibodies, while catalog number 90-1200 is a panel of 6 antibodies against 4 well-known tight junction-associated proteins. Fourteen of our products useful for labeling canine antigens are conjugates labeled with fluorophores or enzymes; 12 of these are mouse monoclonal antibodies and 2 are annexin V conjugates used in apoptosis studies. Annexin V labeled with Alexa Fluor™ 488 and FITC (fluorescein) have catalog numbers A13199, A13202, respectively. We also have 3 kits for detecting apoptosis with canine-derived samples: V13241 and KHO1001 are used in flow cytometry while KHO1021 (ApoTarget™ APO-BRDU Kit) is used for DNA analysis.

In addition to these kits, there are 7 different color- or fluorometric caspase assay kits available (Caspases 3, 8, 9, and a sampler pack for all 3 enzymes) for apoptosis studies with canine and other samples. Samples from canines can also be tested in 4 of our easy-to-use ELISA kits (KAA0021, KHC0061, KHC0064, and KHC1261). We also sell 10 canine hepatocyte-related products, including live and frozen cells derived from beagles. We also have 1 Vesivirus detection kit suitable for testing canine samples, among others (catalog number 44483956).

Specific information about individual antibodies and other products can be found on our website - just search "Primary Antibody Selector Tool" from our home page to find our easy-to-use guide. You can either type "canine" into the search box within the selection guide in the center of the page or click on the canine-specific links under either the Reactivity or Predicted Reactivity columns of the selection tool. If you prefer to browse all our products suitable for use with canine samples, just type either "canine" or "dog" in the main search box at the top of any of our website pages.

Answer Id: E5212

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What are the storage conditions for your Ready-Set-Go! ELISA Sets?

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Answer

All of the reagents are to be stored at 4 degrees C upon receipt, except for the recombinant standards. The recombinant standards MUST be stored at or below -80 degrees C to ensure stability and performance.

Answer Id: E14601

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I am planning to generate a T-REx™ cell line using pcDNA6/TR. Can I perform a western blot using antibodies to TetR to assess whether the cell line is expressing enough of TetR? Do you offer an antibody to TetR?

Product FAQ

Answer

We do not offer an anti-TetR antibody. Even though a western using an anti-TetR antibody can be used to screen out clones that do not express any TetR protein, it would not be the optimal way to screen for functional clones. Functional testing by performing a transient transfection with the lacZ expression control plasmid is recommended for this purpose, followed by picking a clone that shows lowest basal levels of expression of beta-galactosidase in the absence of tetracycline, and highest levels of beta-galactosidase expression upon addition of tetracycline.

Answer Id: E9162

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Is my lipid (Lipofectin™, Lipofectamine™, DMRIE-C, Lipofectamine™ 2000, Oligofectamine™, Cellfectin™ II) supposed to be cloudy?

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Answer

It is normal to see some turbidity and cloudiness in DMRIE-C, Cellfectin™ II and Lipofectamine™ 2000, and the others should be clear. Lipid transfection reagents are sensitive to low temperature; if you put them at temperature below 4 °C they will precipitate or even freeze and lower the efficiency. Most lipids will go cloudy and precipitate upon freezing, and may not be active anymore.

Answer Id: E4000

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I used the iBind™ Flex Western System and have got a large, scattered signal. What could have caused this?

Product FAQ

Answer

Here are possible causes and solutions:

- Protein is overloaded: Reduce load or dilute concentration of sample.
- Poor or incomplete transfer: Repeat blot.
- Primary antibody is too concentrated: Follow the supplier's recommended dilution or determine the optimum concentration by dot blotting.

Answer Id: E11315

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