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Is transfection with mRNA faster? What are standard incubation times? Product FAQ

Answer

Yes, transfection with mRNA results in faster and more immediate translation of protein and therefore, faster expression. We visually see expression of GFP in some cell lines as fast as 90 minutes after transfection. Additionally, transfection of mRNA with Lipofectamine MessengerMAX Reagent also provides prolonged duration of expression (GFP expression lasting for 5 days post-transfection), due to its ability to protect mRNA from degradation during transfection.

Answer Id:: E9035

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Which hybridization oven do you recommend using with an Axiom automated assay? Product FAQ

Answer

We recommend using Model BD 56 from BINDER (https://www.binder-world.com/us/products/standard-incubators/series-bd-avantgardeline/bd-56). This instrument has been validated for use with Axiom automated assays.

Answer Id:: E19573

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Tensile Stimulation of Murine Stem Cell-Collagen Sponge Constructs Increases Collagen Type I Gene Expression and Linear Stiffness Citations & References

  • Authors: Chokalingam, K; Juncosa-Melvin, N; Hunter, SA; Gooch, C; Frede, C; Florert, J; Bradica, G; Wenstrup, R; Butler, DL
  • Journal: Tissue Engineering Part A

Effects of thrombin, hypoxia, and steroids on interleukin-8 expression in decidualized human endometrial stromal cells: implications for long-term progestin-only contraceptive-induced bleeding. Citations & References

  • Authors: Lockwood CJ; Kumar P; Krikun G; Kadner S; Dubon P; Critchley H; Schatz F
  • Journal: The Journal of Clinical Endocrinology and Metabolism
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Specification Sheet: Cytomat 2 C4xx-LIN Standard Series and ToS Series Automated Incubators Dimensions Product Literature

Flyer: Introducing the HCA On-Stage Incubator Product Literature

Standard Wireless Repeater Product Literature

Can I use a heat block for the AutoLys M tube incubation? Product FAQ

Answer

No. The AutoLys M tubes don't fit in a standard 1.5 mL or 2.0 mL heat block. We recommend incubating the tubes in the tube racks in an incubator or oven.

Answer Id:: E16498

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Why are you recommending the overnight incubation for the IVT reaction? Product FAQ

Answer

There are two major benefits for this change in protocol. One benefit is that it provides the option for starting with as little as 1 µg of total RNA using just the standard One-Cycle Labeling protocol. The other benefit is that it incorporates a more convenient and efficient experimental workflow.

Previously, IVT was carried out with 4-5 hours of incubation on the second day of a standard One-Cycle Target Labeling protocol. This made the procedure on the second day rather hectic, since users typically performed the cRNA cleanup (~30 minutes) and fragmentation (~1 hour) after the IVT in order to hybridize their samples on the arrays overnight.

In order to make the workflow more flexible, the new GeneChip IVT Labeling Kit provides a general guideline of using an overnight incubation. However, for customers who prefer the previous protocol, they have the option to spike in additional T7 RNA Polymerase (Ambion, P/N 2085, as described in the product insert) to reduce the IVT reaction time to 4-5 hours.

Answer Id:: E13607

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How do I develop a sandwich ELISA using Antibody Pairs? Product FAQ

Answer

Each Antibody Pair kit contains capture (coating) antibody, biotinylated detection antibody, recombinant standard, and streptavidin-HRP. Other reagents required are listed in the Antibody Pair manual included with the kit, and can also be purchased separately (Antibody Pair Buffer Kit, Cat. No. CNB0011; 5X Assay Buffer, Cat. No. DS98200; etc.). The manual also provides a specific procedure and illustrates an example of a standard curve that can be obtained when the specific procedure is followed.

A general procedure is summarized here:
1) Coat the microplate with diluted capture (coating) antibody overnight at 2-8 degrees C; wash the plate.
2) Incubate the standards or samples in the coated microplate; wash the plate.
3) Incubate diluted biotinlyated detection antibody in the plate; wash the plate.
4) Incubate streptavidin-HRP in the plate for 15-45 min; wash the plate.
5) Incubate the plate with TMB substrate for 10-60 min, and then stop the reaction with Stop solution.
6) Read the microplate at 450 nm.
We recommend determining optimal buffer formulations, concentrations, and incubation times for individual applications.

Answer Id:: E12616

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How do I develop a sandwich ELISA using Cytosets? Product FAQ

Answer

Each CytoSets contains capture (coating) antibody, biotinlyated detection antibody, standard and Streptavidin-HRP. Other reagents required are listed in the CytoSets information sheet included with the kit and can be purshased from us separately (Antibody Pair Buffer kit CNB0011, 5x Assay Buffer DS98200, etc.). The information sheet also provides a specific procedure and illustrates an example standard curve which can be obtained when the specific procedure is followed. A general procedure is summarized here:

1) Coat the microplate with diluted capture (coating) antibody overnight at 2-8 degrees C; Wash the plate
2) Incubate standards or samples with the coated microplate; Wash the plate
3) Incubate diluted biotinlyated detection antibody with the plate; Wash the plate
4) Incubate Streptavidin-HRP with the plate for 15-45 minutes; Wash the plate
5) Incubate the plate with TMB substrate for 10-60 minutes and stop the reaction with Stop solution
6) Read microplate at 450 nm.

Investigators are advised to determine optimal buffer formulations, concentrations and incubation times for individual applications.

Answer Id:: E5138

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My gel stained with Pro-Q Emerald 300/488 Glycoprotein Gel Stain is showing strong staining of non-glycosylated proteins, including more than four bands in the CandyCane marker lane. What can I do to improve specificity? Product FAQ

Answer

Over-oxidation with periodate during the step 2.5 Oxidizing Solution incubation will cause strong non-specific staining of non-glycosylated proteins. Do not incubate standard mini-gels or blots longer than 30 min, or large format 2D gels longer than one hour or use more than the recommended concentration of periodate.

Answer Id:: E11297

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Can the reaction time go beyond 2 hours for protein synthesis in the Expressway system? Product FAQ

Answer

The standard reaction time is 2 hours. However, increasing the time to 4 hours may increase the yield of protein. For less soluble proteins, this longer incubation should be carried out at ambient temperature.

Answer Id:: E9653

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After I added the chromogen reagent to the plate, I incubated the ELISA as suggested in the manual, but the A450 of the highest standard was higher than what my plate reader can read. What should I do? Product FAQ

Answer

Our customers use a wide variety of plate readers. Some of these can't read absorbances higher than 2 AUFS (Absorbance Units Full Scale), while others can't go beyond 3 AUFS, for example. If you read your ELISA plates after 30 minutes of incubation at room temperature and 1 or 2 A450 values are off-scale, you can shorten the incubation time. For example, some customers find that 20 minutes is the ideal incubation time because the ambient temperature in their lab is higher than approximately 2 degrees F (22 degrees C). In this case, higher temperatures increase the rate of the HRP-driven ELISA. Conversely, if the A450 values you get are not high enough, you can increase the incubation time accordingly.

Answer Id:: E12629

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Can you summarize the sulfhydryl quantitation protocol for Thermo Scientific Ellman's Reagent? Product FAQ

Answer

Sulfhydryl groups may be estimated in a sample by comparison to a standard curve composed of known concentrations of a sulfhydryl-containing compound such as cysteine, preferrably at a working range of 0.1 - 1.0 mM. A volume of 250 µl is required for the sample and standard, mixed with Ellman's Reagent Solution and Reaction Buffer, then incubated for 15 minutes at room temperature. Absorbance is read at 412 nm.

Answer Id:: E13372

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