Search
Search
View additional product information for WesternDot™ 625 Western Blot Kits - FAQs (W10132, W10142)
38 product FAQs found
Any dye in the 625 nm emission range should work. For example, you can use the WesternDot 625 Western Blot Kit.
We also offer our White-Light Conversion Screen (Cat. No. 4473061), which converts blue light emitted by the blue-light transilluminator or UV light emitted by the UV-light transilluminator to white light. This conversion screen is compatible with multiple protein stains including SimplyBlue SafeStain, SilverQuest silver, and Coomassie blue stains.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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 antige: 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.
- WesternDot reagents have been frozen: Qdot probes will irreversibly aggregate at freezing temperatures
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
If there is a large amount of precipitate, it likely indicates that the reagent has been frozen and should be discarded. Qdot probes will irreversibly aggregate at freezing temperatures. The formation of a small amount of precipitate during storage at 2-8 degrees C is normal. We recommend spinning the WesternDot reagent tube down briefly in a microcentrifuge with every use to remove any minor precipitate that has formed during storage and use only the supernatant.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
There are a number of endogenous biotinylated proteins in cell lysate samples that will also be detected with streptavidin WesternDot conjugates. Endogenous biotinylated proteins can be confirmed by incubating a control blot in only the streptavidin WesternDot conjugate without the primary and secondary antibody. These endogenous biotinylated protein bands will be consistent in every lane containing the same cell lysate type and can serve as internal loading controls or can be blocked prior to western detection using the reagents in the Endogenous Biotin Blocking Kit (Cat. No. E21390).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
WesternDot signals can fade when bound to dried PVDF membranes. For archiving stained blots or imaging after more than 24 hours, we recommend using nitrocellulose membranes.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Many factors may reduce signal intensity. One major factor is the primary antibody. Primary antibodies from different sources can have very different affinities to the target antigen. If weak signal is observed, try a primary antibody from a different vendor and /or from different host species, as this can help to improve signal intensity.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Both nitrocellulose and PVDF membrane can be used. For better results, we recommend using nitrocellulose membrane to minimize background from autofluorescence.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
WesternDot detection reagents are stable for at least 6 months from the date of receipt when stored at 2-8 degrees C, We do not recommend freezing them.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
This may be possible, but has not been validated. A biotinylated probe should be detected with any of our streptavidin labeled Qdot probes (such as our Qdot 625 streptavidin conjugate, Cat. No. Q22063) using standard northern or Southern blotting protocols.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The sensitivity of WesternDot detection reagents and the far-red dyes are all similar to or better than ECL detection. Both types of reagents use similar staining protocols and can be archived on dried blots. Also, WesternDot reagents and far-red dyes are both directly linked to the primary antibody target protein, so the resolution of the stained protein band is very sharp, rather than the diffuse edges around a protein band that is seen with chemiluminescent and chromogenic detection via an enzyme/substrate. The advantage of the WesternDot reagents over far-red fluorescent dyes is that UV-blue excitation wavelengths are so far removed from the emission wavelengths that there is virtually no autofluorescence background from the blot membrane or blocking proteins, giving much better signal to noise and thus very high contrast images. Far-red fluorescent dyes are excited close to their emission wavelengths, so there will be some autofluorescence background from the blot membrane and blocking proteins. WesternDot detection reagents are much more photostable than fluorescent dyes and do not require protection from light during staining or handling. WesternDot detection reagents require a UV or blue light excitation source commonly available on most gel and blot imagers and do not require the purchase of a specialized imager. WesternDot 625 reagents use the same excitation sources and emission filters as ethidium bromide-stained gels.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
This is not recommended, as Qdot probes used to make the WesternDot conjugates are significantly larger than IgGs. Qdot probes are estimated to be about 1.5-2 million Da (similar in size to an IgM), so permeability through the gel matrix is going to be much lower than for an unlabeled antibody or even an HRP- or alkaline phosphatase-labeled antibody. It may be possible in a very low percent acrylamide gel with some optimization, but sensitivity will be less than on a membrane.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
No, the Li-COR Odyssey imagers use excitation wavelengths of 685 nm and 785 nm. For optimal sensitivity, you need to use an instrument that has UV or blue light excitation. WesternDot reagents have weak excitation near their emission peak, so you may see some signal, but sensitivity will be much lower than for far-red fluorescent dyes.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
When imaging a WesternDot conjugate-stained blot, the blot should be placed with the protein side toward the light source. On a UV transilluminator, this means that the blot should be placed face down.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The brightest signal and lowest sensitivity are obtained when exciting in the UV range, but imagers equipped with a blue light source (e.g., 450 nm or 473/488 nm) can also be used.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
No, blots can be imaged wet or dried. For long-term stability of signal, blots should be kept dry.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The photostability of WesternDot reagents enables long-term storage of dried blots without loss of signal intensity.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The photostability of WesternDot reagents enables taking multiple images without loss of signal intensity.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Any basic gel or blot imager with UV or blue light excitation and an ethidium bromide filter can be used with for single-color detection of WesternDot 625 conjugates. The E-Gel imager with the UV or blue light base and the orange filter can be used for detecting single color WesternDot 585 or WesternDot 625 conjugate-stained blots. For two or three-color multiplexing, a GE Healthcare ImageQuant LAS 4000 includes filter sets suitable for imaging WesternDot conjugates in the 585, 625, 655, and 800 colors. Other specific imager and filter combinations for all the WesternDot reagents can be found in Table 2 of the Western Dot Secondary Antibody Conjugates manual (http://tools.thermofisher.com/content/sfs/manuals/westerndot_secondary_ab_conj_man.pdf).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Yes, western blot processing instruments work well with WesternDot reagents.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Yes, it is possible to multiplex detection with WesternDot reagents and WesternBreeze CDP-Star substrate, WesternBreeze BCIP/NBT and ECL reagents. WesternBreeze CDP-Star is the preferred detection reagent for this application. Simply co-incubate in the primary antibodies and then in the secondary antibodies following the WesternDot detection protocol. The CDP-Star chemiluminescent signal is not detected with UV excitation and emission filters used for WesternDot imaging, so the WesternDot signal can be imaged before CDP-Star substrate incubation or afterwards on wet or dried blots. WesternBreeze BCIP/NBT and ECL signals will be detected in the WesternDot image, so the WesternDot signal must be imaged first on a wet blot before addition of these detection reagents. Here is a link to a more detailed protocol (https://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/support/bioprobes/bioprobes-63.par.51550.file.dat/bioprobes-63-westerndot.pdf).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Yes, blots can be stripped and reprobed with WesternDot reagents. We found that in general, the WesternDot conjugate was removed easier than standard dye or protein-labeled secondary conjugates and that the primary antibody was the hardest to remove. You should be able to use any stripping solution that is strong enough to remove the primary antibody. We have obtained good results with Restore and Restore Plus stripping buffers following their protocol at room temperature. It may take heating at 50 degrees C to fully remove the primary well. The multiplexing capability of WesternDot reagents enables simultaneous detection of multiple proteins using appropriately compatible primary antibodies and WesternDot reagents, so that stripping and reprobing is no longer required.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The process for multiplexed protein detection is the same as for single protein detection. After transferring proteins onto PVDF or nitrocellulose membranes and blocking, incubate the blot in a mixture of primary antibody solution for each protein target followed by detection with a mixture of WesternDot secondary antibody conjugates against each primary antibody. For example, to do a three-color multiplexing of Akt, phospho-Akt and GAPDH, incubate the blot in a mixture solution of rabbit anti Akt antibody, mouse anti phospho-Akt and chicken anti GAPDH, followed by detection with a mixture of WesternDot 605 goat anti rabbit IgG, WesternDot 655 goat anti chicken whole IgG and WesternDot 800 goat anti mouse IgG.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
No, the WesternDot staining solution should be used as soon as it is prepared and can only be used once. Qdot probes are not stable long-term diluted in buffer.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
No, it is not necessary. Qdot probes are photostable under normal room light illumination, so blots can be incubated and dried without covering to protect from light.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Tween 20 is a detergent commonly used in western blot buffer formulations. For WesternDot secondary conjugates, we found the use of Tween 20 in buffers to be suitable for nitrocellulose membrane blots, but not necessary for desirable results. Because Tween 20 leads to high background on PVDF membrane, it is not recommended for use with WesternDot secondary conjugates in combination with PVDF. If PVDF membranes are used, Tween 20 should be omitted from all the buffer formulations.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
For best results, refer to the vendor's suggested incubation time, or optimize incubation time. Generally, the primary incubation is carried out for one hour at room temperature, or overnight at 4 degrees C. We recommend an incubation time of 1 hour at room temperature for WesternDot secondary antibody conjugates.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The recommended WesternDot secondary antibody conjugate working dilution is 1:500. Each vial of WesternDot secondary antibody conjugate contains sufficient material for twenty-five western blots at this working dilution.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The ideal working concentration can vary between different primary antibodies. For best results, one should refer to the antibody vendor's suggested conditions, or optimize the working concentration for a primary antibody prior to use.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
No single buffer formulation works best for every protein/antibody:
- 2% casein, 5% non-fat dry milk, or 1/2X fish serum (e.g., Fish Serum Blocking Buffer) 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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
For western blots, where proteins are freshly transferred from SDS-PAGE gels to nitrocellulose or PVDF membranes, washing the membranes twice for 5 mins with 20 mL of pure water is recommended to partially remove gel and transfer buffer components and weakly bound proteins. The membranes are then ready for the WesternDot immunodetection protocol.
Alternatively, the washed membranes may be dried on a clean piece of filter paper in open air, by a stream of slightly warm air or under an infrared lamp. Properly dried membranes can be stored in a closed container at 4 degrees C for several days depending on the antigen loaded. Water-washed and dried nitrocellulose membranes are ready for the WesternDot immunodetection protocol. However, water-washed and dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 minutes before proceeding to the WesternDot immunodetection protocol.
For Native-PAGE Western blots, a drying step, performed before any washing steps, is recommended to improve protein binding to the membrane. Once dried, nitrocellulose membranes should be washed twice with 20 mL water for 5 minutes before proceeding to the WesternDot immunodetection protocol. Dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 minutes before proceeding to the WesternDot immunodetection protocol.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
All standalone WesternDot reagents are provided with sufficient material to stain 25 Mini gel blots. The WesternDot625 Goat anti-mouse and goat anti-rabbit kits (Cat. Nos. W10132 and W10142) are provided with sufficient reagents to stain 20 Mini gel blots.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Yes, they can be purchased as Cat. Nos. B2763 and B2770, respectively.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Yes, it can be purchased as Qdot 625 Streptavidin Conjugate (Cat. No. Q22063, 50 µL or Cat. No. A10196, 200 µL).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
WesternDot 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 WesternDot reagents to the primary antibody target protein, rather than the diffuse edges around a protein band seen with enzyme-based detection methods
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
WesternDot reagents have similar or slightly better sensitivity compared to ECL detection. The sensitivity is in the picogram range.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
WesternDot reagents are whole IgG (H+L) conjugates of our popular secondary antibodies and select monoclonal primary antibodies, or streptavidin conjugates of our red and far-red fluorescent Qdot probes, Qdot 585, Qdot 625, Qdot 655 and Qdot 800, for western blotting applications. Qdot probes have narrow and symmetrical emissions, minimizing overlap with other emission colors, producing less bleed through into adjacent detection channels and allowing multiple colors to be used simultaneously. They are equally optimally excited with any UV or blue light sources commonly available on gel and blot imagers. Furthermore, Qdot probes are extremely photostable, making it possible to take multiple images and store dried blots for months with minimal loss of fluorescent signal.
For more information on our Qdot technology, see the following link:
https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/ultrasensitive-detection-technology/qdot-nanocrystal-technology.html
http://www.thermofisher.com/us/en/home/brands/molecular-probes/key-molecular-probes-products/qdot/technology-overview.html
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.