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View additional product information for BenchPro™ 4100 Western Card - FAQs (WP1001)
34 product FAQs found
Yes, the blot will be held in the last reagent programmed, regardless which reagent that is. For the blot to be left in wash buffer, just make wash the last step instead of rinse.
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Here are some recommendations that may help:
- Use a PBS-based Blocking reagent and do not use Tween 20 or BSA.
- Make sure that the Primary and Secondary antibodies are freshly diluted in 0.01% Tween 20 in PBS
- For the last 2 washes, use only PBS to get rid of the Tween 20, thus avoiding background.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
We would recommend shutting off the power and restarting the instrument.
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This could be due to the following reasons:
- AC power cord is not connected or incorrect cord was used.
- Fuse has blown.
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Here are possible causes and solutions:
- The Western Card may not be in the right position: Pull it out and insert it again to make sure it is inserted all the way down until you hear a click sound.
- Solution intake tips could be clogged: Check your solutions, especially any milk blocking buffers, to see if there are large particles present that could be blocking the tips.
- Defective cards: Try a new card, or a card from a box with a different lot number if possible. If you suspect a problem with your cards, please contact Technical Support at techsupport@thermofisher.com.
- Check instrument pressure and vacuum value: If numbers are significantly below 248 kPa for instrument pressure and below 48 kPa for vacuum, there could be a possible hardware issue. Please contact Technical Support at techsupport@thermofisher.com .
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are possible causes and solutions:
- Film overexposed or became wet during exposure: Decrease exposure time or allow signal to further decay. Prevent leakage of solutions by encasing membrane in transparency film and blotting excess substrate from edges before exposure.
- Short blocking time or washing time: Perform each step for the specified amount of time.
- High concentration of primary and/or secondary antibody: Determine optimal antibody concentration by performing a dot blot. Decrease antibody concentration as necessary.
- Membrane, solutions, trays, or vials are contaminated: Use clean glassware and purified water to prepare solutions. Rinse the tray thoroughly with purified water. Although all reagent bottles and vials can be reused after washing with mild detergent and rinsing with deionized water, we do not recommend reusing the 25 ml vials to prevent any reagent cross-contamination. Wear clean gloves at all times. Use forceps when handling membranes.
- Protein is overloaded: Reduce load or dilute the sample.
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Here are possible causes and solutions:
- Detection step missed or detection reagents not working: The detection step is not included in the instrument protocol. After the instrument protocol is complete, perform the detection step using your standard detection reagents and protocol manually. Make sure the detection reagents are functional.
- Insufficient incubation with detection reagent: Remove the blot from the detection reagent when signal-to-noise ratio is acceptable.
- Incorrect order of reagents added: Be sure to add the reagents in the order described on page 13 of the manual (https://tools.thermofisher.com/content/sfs/manuals/benchPro_4100_man.pdf) to ensure all steps are performed correctly.
- Poor or incomplete transfer: Make sure transfer apparatus and membrane sandwiches are assembled correctly. Use appropriate transfer times. After blotting, stain membrane to measure transfer efficiency.
- Protein of interest ran off the gel: Use positive control and/or molecular weight marker to match gel separation range to size of protein being blotted. After blotting, stain membrane to measure transfer efficiency.
- Sample was too dilute: Load a larger amount of protein onto the gel.
- Poor retention of proteins or protein weakly bound to membrane: Ensure that transfer buffer contains 10-20% methanol. Use membranes with appropriate binding capacity.
- Inactive or overly dilute primary or secondary antibody: Determine antibody activity by performing a dot blot. Increase antibody concentration as necessary.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are possible causes and solutions:
- Bottles have caps: Make sure that the reagent bottles and vials are uncapped when placed in the reagent tray.
- The card is not oriented correctly: Insert the card into a slot in the BenchPro 4100 Card Processing Station such that the tips are inserted into the reagent bottles and vials placed in the reagent tray. Use the Card Alignment Guide to ensure the card is placed correctly in the slot. When the card is inserted correctly in the slot, you will hear a hard click.
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We recommend making sure that you have added the card, reagents, and reagent bottles in the correct order prior to starting the protocol. If the run is aborted after the run has begun, you need to restart the run. Do not reuse the card, if the run is aborted within a few minutes of starting the run.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Please refer to the list of error messages and actions to take on page 30 in the manual (http://tools.thermofisher.com/content/sfs/manuals/benchPro_4100_man.pdf).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are possible causes and solutions:
- AC power cord is not connected. Check AC power cord connections at both ends. Use the correct cords.
- Fuse has blown. Replace the fuse (see page 31 in the manual http://tools.thermofisher.com/content/sfs/manuals/benchPro_4100_man.pdf). If the problem still persists after verifying that correct power cord is used and the fuse is replaced, contact Technical Support at techsupport@thermofisher.com.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Functionally, steps and sub-steps will perform the same. However, you cannot program a sub-step without first programming a step (an error message will display if you try). If you select 'step' and then immediately select 'sub-step', the 'step' will default to 1 min for the 'minutes' but no 'times' will be saved and the protocol that is saved will display the entry as a 'step'. This can be seen when the protocol is viewed again after it is saved.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The BenchPro 4100 allows for overnight incubation by increasing the time of the primary antibody incubation step to several hundred mins. The rest of the reagents need to be loaded in the instrument from the beginning, though, as there is no way to halt the run the next day, remove the card to load new reagents, then continue the same protocol with the same card as the previous day. If there is concern about reagents sitting overnight at room temperature, the instrument can be run in a cold room or deli cooler.
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Unfortunately, the abort does not send the reagents back to where they came from; they randomly drain through the card.
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About 0.5-1 mL of each buffer will remain in the card and tips after the run.
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The faster program will generally give sensitivity in detection that is 70-80% of the standard WesternBreeze immunodetection protocol.
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The fuse is 250 V, 1.25 A. We do not offer it as a standalone product, but it can be purchased from electronics vendors such as RadioShack.
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No, but please ensure that the reagent bottles and vials have been loaded in the same tray row as the cards that will be used. The unit automatically detects which slots contain cards and only the slots containing cards are functional during processing.
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We do not have any validated protocols for this, but we suggest covering the Western Card with foil or running the experiment in a dark room if possible.
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Yes, the processor can be programmed to return reagents back into the same vials after use.
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The operating temperature range is 4-40 degrees C. So the instrument can be operated in the cold room or incubator if needed. However, we have only tested for a few days in the cold room.
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No, the cards are not meant to be reused. The instrument detects used cards by a color indicator spot at the lower right hand corner of the card that turns red after the card is used.
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There is no need to use all 4 cards at a time. You can use one card or 2-4 cards at a time.
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8.5 cm x 8.5 cm is the maximum membrane size that can be processed.
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Yes, as each card is filled from a separate set of vials.
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The instrument has the capacity to process 1-4 Western Cards. Each card could process 1 or 2 membranes at a time, placed back to back. Therefore, the system has the capacity to simultaneously process up to 8 membranes in each run.
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The BenchPro 4100 Card Processing Station is pre-programmed with three of the most popular western protocols: two for Western Breeze immunodetection and one for standard ECL detection. These protocols are programmed to return the primary antibody to the original vial. Additionally, 17 more custom protocols can be stored in the system's memory. The custom protocols can be programmed to return any of the reagents back to the original container, if desired.
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We do not offer the 6 ft Drain tubing for Auto Drain. It is 1/4 inch OD x 1/8 inch ID and you may purchase it from http://www.mcmaster.com/#catalog/119/136/=q5ikda.
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The 25 mL Reagent Vials can be purchased separately as a set of 50 vials/package (Cat. No. WP3001). However, we do not offer the Reagent Bottles as standalone products. You may purchase them from Cole Parmer:
- 250 mL Reagent Bottles (Set 1) (Cat. No. EW-06019-76) (http://www.coleparmer.com/Product/Bottles_Squre_HDPE_250ml_10_pk/EW-06019-76
- 125 mL Reagent Bottles (Set 2) (Cat. No. EW-06254-20) (http://www.coleparmer.com/Product/Thermo_Scientific_Nalgene_Sterile_PETG_Media_Bottles_125_mL_48_box/EW-06254-20)
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Clean the surface of the BenchPro 4100 Card Processing Station with a damp cloth. Do not use harsh detergents or solvents to clean the unit. Rinse the reagent tray assembly and waste basin with water after each use. If bottles and vials are to be reused, clean with mild detergent and rinse thoroughly with ultrapure water. Do not autoclave trays or reagent bottles and vials. Although all reagent bottles and vials can be reused after washing with mild detergent and rinsing with deionized water, we do not recommend reusing the 25 mL vials to prevent any reagent cross-contamination.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Yes, the BenchPro 4100 Card Processing Station has been discontinued as of December 31, 2014. The current BenchPro 4100 Western Cards (https://www.thermofisher.com/order/catalog/product/WP1001) and Reagent Vials (https://www.thermofisher.com/order/catalog/product/WP3001) will continue to be available for purchase. We offer the new iBind Western Device (http://www.thermofisher.com/us/en/home/life-science/protein-expression-and-analysis/western-blotting/western-blot-detection/ibind-western-system.html) as an alternative system for automated western blot processing.
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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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Yes, western blot processing instruments work well with WesternDot reagents.
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