My DETACHaBEAD™ beads look cloudy, is it normal?

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Answer

Yes, the cloudy appearance is normal for DETACHaBEAD™ beads because the protein content is very high and the protein can precipitate. This is not bacterial contamination and does not affect the recovery or viability of the isolated and released cells. Mix the solution thoroughly before use.

Answer Id: E12070

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I used the Invitrogen™ Semi-Dry Blotter and the transfer efficiency was very poor. Can you please help me troubleshoot?

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Answer

Here are possible causes and solutions:

- Voltage is too low: 1 mm thick polyacrylamide gels (Mini and Midi Gels) should be transferred at 20 V, E-PAGE™ Gels at 25 V (approximately 15 V/cm field strength).
- Power supply is inappropriate for semidry transfer: Some power supplies will shut off or blow a fuse when run at the conditions required for semi-dry transfer. Semi-dry transfer requires low voltage (20 V) and high current. Check with the manufacturer of the power supply to determine whether it is appropriate for semi-dry transfer.
- Transfer was performed for too short a time: Increase the amount of time for transfer. Typical transfer times range from 30 to 60 minutes.
- Transfer sandwich was assembled in the wrong order: The Invitrogen™ Semi-dry Blotter is configured with the cathode on the top, and anode on the bottom. This results in a downward transfer of proteins from the gel onto the membrane. Follow the instructions carefully when assembling the transfer sandwich.
- The pH of the transfer buffer is too close to the isoelectric point of the protein: The transfer buffers should be at the optimal pH if prepared as described in this manual. Do not adjust the pH with acid or base as this will increase the conductivity of the buffer and result in higher current during transfer.
- Too much methanol is present in the transfer buffer: Reducing the amount of methanol can help elute proteins from the gel, but can reduce binding to nitrocellulose membranes.
- High-percentage gels restrict transfer: Higher percentage acrylamide or crosslinkers can restrict elution of proteins. Use the lowest percentage acrylamide possible to separate your proteins.
- Puddles of buffer were present on the plates, allowing the current to bypass the stack: Always clean up the lower plate before closing the lid of the transfer apparatus. Do not squeeze the stack excessively, as this removes transfer buffer from the blotting paper and also creates puddles that the current can pass through.

Answer Id: E11609

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What are the main reasons for performing 2D gel electrophoresis of proteins?

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Answer

The main advantages of performing 2D gel electrophoresis of proteins and applications used for are listed below:

Advantages:

*Simultaneous separation of hundreds to thousands of proteins
*High capacity with superior resolution
*Compatible with further analysis by MS for protein identification and sequencing
Ability to separate and analyze low-abundance proteins

Applications:

*Comparative proteomics: identifying and analyzing differences between complex mixtures of proteins
*Protein profiling, biomarker discovery
*Separation and analysis of protein variants and isoforms

Answer Id: E10623

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I use TesR™ Medium as a PSC culture medium. Is PSC Definitive Endoderm Induction Medium compatible with this medium?

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Answer

Optimal results are obtained by using Essential 8™ Medium as a pluripotent stem cell medium, however mTesR1™ or TeSR™-E8™ can be used as substitute, if desired.

Answer Id: E9385

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How should I prepare the starting cellular material for isolation using Dynabeads™ magnetic beads?

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Answer

It is very important to properly homogenize the cells before use. Homogenization may be easier for some samples such as liver than for other samples such as yeast. If nuclei are broken during homogenization, DNA will be released and may cause clumping of organelles. Clumping of organelles in the unpurified cellular material will make immunoisolation difficult.

Answer Id: E6148

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How much is 10 GB of storage on the Cloud?

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This is roughly the amount of data generated by the analysis of 140 Appiled Biosystems™ OpenArray™ plates.

Answer Id: E10234

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What’s the difference between the T4 DNA Ligase and the ExpressLink™ T4 DNA Ligase?

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Expresslink™ T4 DNA Ligase allows for faster ligation times and ligation at room temperature. For TA cloning, the ligation time is 15 minutes while the original T4 DNA ligase needed a minimum of 4 hours to overnight incubation. For blunt cloning, the ligation time with the Expresslink™ T4 DNA Ligase is reduced to 5 mins from 1 hour with the T4 DNA Ligase. The performance of these two ligases is similar, with a 80% cloning efficiency with our control PCR inserts using the above mentioned minimum ligation incubation times/temperatures.

Answer Id: E6662

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Can you offer tips to increase cell yields when using DETACHaBEAD™ beads?

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Answer

Here are some recommendations:

- Mix the DETACHaBEAD™ solution thoroughly before use.
- The most critical parameter for a good yield is mixing of the beads with the sample during the incubation step. We recommend using a mixer or rotator (e.g., HulaMixer™ Sample Mixer (Cat. No. 15920D)) that keeps the tube continuously in motion, but in such a way that the sample stays in the bottom part of the tube to avoid drying out of the beads.
- Reduce the incubation time during cell isolation (lower binding affinity can increase release efficiency).
- After incubation with DETACHaBEAD™ beads, pipette the bead-bound cells up and down at least 10 times before applying to the magnet to get as many beads off the cells as possible. Too vigorous pipetting can lower the viability.
- Avoid unnecessary delays during the protocol.

Answer Id: E12071

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What is the mechanism of exosome formation?

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Answer

Exosomes are classically described as vesicles originating from the endosomal compartment through fusion of multivesicular bodies with the plasma membrane. They are a part of a larger family of vesicles secreted by cells, including microvesicles, ectosomes, and shed particles, which originate by direct budding from the plasma membrane. It is extremely challenging to separate these entities using currently available techniques and instruments due to overlap in their size, density, and overall similar composition.

Answer Id: E7445

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I'm trying to express my protein in vitro and am getting low to no protein yield, whereas my control reaction is producing protein. What should I do?

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Answer

Please review the following suggestions:

- Check the sequence of your vector (ATG initiation codon, in frame, etc.).
- If working with a N- or C-terminal tag, the tag may be affecting the RNA structure and lowering translation levels. Try moving the fusion tag or the other terminus.
- Ensure that your DNA template is pure, and not contaminated with ethanol, sodium salt, ammonium acetate, or RNases.
- Do not purify your DNA from an agarose gel, as this can inhibit the reaction.
- We recommend using 10-15 μg of template DNA in a 2 mL protein synthesis reaction. If you are expressing a large protein, increase the amount of DNA template used in the protein synthesis reaction to 20 μg.
- Ensure that you are using a thermomixer or incubator with shaking, as opposed to a non-shaking incubator or water bath for the reaction.
- Multiple feeding steps can further improve the protein yield. Instead of doing one feeding at 30 min of the initial reaction, you can feed the reaction multiple times with smaller volumes of feed buffer to the sample more frequently (i.e., 0.25 mL feed buffer to 1 mL sample every 45 min over 3 hours) after initiating protein synthesis.

Answer Id: E9680

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I performed a semi-dry transfer using the Invitrogen™ Semi-Dry Blotter and there was insufficient binding of proteins to the membrane. Can you offer some tips?

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Answer

Here are possible causes and solutions:

- Air spaces are interfering with contact between the gel and the membrane: Roll the membrane with a blotting roller (or a clean test tube or pipet) before placing the gel on the membrane, and then remove any air bubbles between the gel and membrane with a blotting roller or a wet gloved finger. Transfer will not occur where the gel is not in contact with the membrane.
- Electrophoretic conditions were incorrect or not ideal: Running conditions, sample preparation, percentage acrylamide, and many other variables can affect the migration and resolution of proteins. Please review your electrophoresis conditions.
- Under- or over-compression of gel: Follow the compression guidelines in the manual (http://tools.thermofisher.com/content/sfs/manuals/Invitrogen™_semidry_blotter_man.pdf). Too little compression can allow proteins to migrate between the gel and membrane, causing protein band smearing. Too much compression can distort the gel.

Answer Id: E11610

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What are ampholytes and do you offer them?

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Answer

Ampholytes are molecules that contain both acidic and basic groups (and are therefore amphoteric) and will exist mostly as zwitterions in a certain range of pH. The pH at which the average charge is zero is known as the molecule's isoelectric point. Ampholytes are used to establish a stable pH gradient for use in isoelectric focusing. In gels containing ampholytes, a linear pH gradient is built up when an electric field is applied. The ampholyte molecules carry a net charge and thus migrate in the electric field between the electrodes as long as they reach the position of corresponding pI. They will then stop moving and form small plateaus (stationary stacks). Commercially available ampholytes are a mixture of synthetic molecules whose individual pI values cover a preselected pH range. Thus one can purchase carrier ampholytes spanning either a wide pH range (e.g., pH 3-10) or a narrow range (e.g., pH 7-8).

We offer ZOOM™ Carrier Ampholytes that are small, soluble molecules with both positive and negative charge groups. They sort at their relative positions based on their isoelectric points in an electric field, setting up a pH gradient. Carrier ampholytes help stabilize the pH gradient and current in IPG strips and aid in protein solubility, resulting in reproducible IEF resolution.

Answer Id: E10624

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At what seeding density should I seed PSCs prior to differentiation?

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Answer

The optimum range for seeding density is 0.01 × 10e6-0.04 x 10e6 cells/cm2. For example seed 0.09 x 10e6 - 0.38 x 10e6 cells per well of a 6-well plate.

Answer Id: E9386

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Is the EBNA-1 gene expressed in 293-EBNA Cells full-length?

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Answer

Yes, the EBNA-1 gene in this cell line is full length.

Note: 293-EBNA cells are no longer available for purchase.

Answer Id: E4327

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What is the difference between the “R” buffer and the “E" or "E2” buffers for Neon?

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Answer

The “R” buffer is used to resuspend the cells prior to transfection (“R” for Resuspension”). The “E or E2” buffers are the electrolytic buffers used for electroporation. “E or E2” buffers are added to the Neon tube prior to electroporation

Answer Id: E5472

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