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Regulatory statements vary by product. Please see product-specific literature to determine product use.

Can I use a Dounce homogenization procedure in conjunction with the Mitochondria Isolation Kit for Cultured Cells (Cat. No. 89874)? Product FAQ

Answer

Yes, the kit offers an optimized Dounce homogenization procedure, which results in two-fold greater mitochondri recovery compared to reagent-based method.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Answer Id: E13225

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What reactive groups do you offer for your surface-activated Dynabeads magnetic beads? Product FAQ

Answer

We offer tosyl-, epoxy-, carboxylic acid-, and amine-activated Dynabeads magnetic beads. See our bead comparison on the following link (https://tools.thermofisher.com/content/sfs/brochures/Surface_Activated_Dynabeads.PDF).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E13039

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My protein did not immunoprecipitate. What went wrong? Product FAQ

Answer

Make sure that the sample contains enough antigen by verifying its expression and/or lysis efficiency of the lysate by SDS-PAGE or western blotting.

Secondly, make sure the antibody solution does not contain amines or carrier proteins which could compete with the antibody for coupling to the resin. You can verify the antibody coupling by monitoring the flow-through and wash fractions (i.e., measure the absorbance at 280 nm or analyze by SDS-PAGE).

Finally, it is possible that a component in the IP Lysis/Wash Buffer interfered with antibody-antigen binding. To prevent this, perform the IP and washes using 1X Tris-buffered saline.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E13051

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What is the detection limit when using Dynabeads magnetic beads for immunoprecipitation (IP)? Product FAQ

Answer

Answering this question is not straightforward. It will depend on the detection method. When using HRP (horseradish peroxidase)-based detection system or radioactivity in combination with a good antibody, very little target is required. More target is required when using an AP (alkaline phosphatase)-based detection system. When a sensitive detection system is used, detection will most likely be in the nanogram range. In some cases, pictograms of target can be detected.

Find additional tips, troubleshooting help, and resources within our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.

Answer Id: E13015

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What are the Gauss levels for the DynaMag-2 and DynaMag-Spin Magnets? Product FAQ

Answer

The DynaMag-2 Magnet is 3,500-3,700 minimum Gauss while the DynaMag-Spin Magnet is 3,000-3,500 minimum Gauss.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E13047

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What should I do to increase the coupling efficiency of antibodies to the Pierce NHS-Activated Magnetic Beads? Product FAQ

Answer

-Make sure all primary amine-containing buffer components of the buffer like Tris or glycine are completely removed by dialysis, desalting, or the use of the Pierce Antibody Clean-Up Kit (Cat. No. 44600) before coupling to magnetic beads
-Make sure the antibody is mixed with the NHS-beads IMMEDIATELY after washing the beads. A delay may cause hydrolysis of the NHS groups on the resin which leads to low coupling efficiency.
-Finally make sure the pH of the non-amine-containing coupling buffer used for coupling is between pH 7-9

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E13055

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My interacting protein was not isolated during the pulldown; what should I do? Product FAQ

Answer

Here are some reasons why the immobilized “bait” partner may not pull down the interacting “prey” protein and some tips to improve the pulldown:

-It is possible that the prey protein is degraded: in this case, include protease inhibitors and or phosphatase inhibitors (see examples) in the lysis buffer. Also use fresh lysate or lysate frozen at -80 degrees C.
-If the interaction between bait and prey is weak or transient, such that the stringent wash conditions disrupt the interaction, you can reduce the number of washes and ionic strength of wash buffer.
-If the prey protein is expressed at a low level, apply more protein (prey) sample, increase amount of bait protein and incubation time.
-Test to see if any cofactor is essential for the interaction being studied and include this in the incubation.
-It is also possible that binding or sample preparation conditions were insufficient to maintain or allow protein interactions. In this case, try alternative buffers for sample preparation, binding or washing procedures.
-Excessive labeling (e.g., biotinylation) of the bait could sterically hinder binding to the prey protein. In this case, reduce molar excess of the biotinylation reagent used for labeling or use a reagent that targets a different functional group.


Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E13067

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What should I do if I get nonspecific binding to Dynabeads Protein A or Protein G magnetic beads? Product FAQ

Answer

Here are a few suggestions to try:

-Use more stringent conditions for washing
-Add a non-ionic detergent (Tween-20 or Triton X-100) to the washing buffer, at concentrations between 0.01-0.1% (v/v)
-If the beads are blocked before precipitation, add the same blocker to the washing buffer
-Increase the number of washing steps
-Prolong the duration of the washing steps and/or add a soak step
-Decrease the incubation time between the beads and the sample
-Try the indirect IP method (add the antibody to the sample, incubate, then capture the immune complexes with the beads)
-Decrease the antibody concentration
-A pre-clearing step may be performed to remove molecules that non-specifically bind to the Protein A/Protein G and/or the beads themselves

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E13059

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How should the Cas9/gRNA ribonucleoprotein (RNP) complex be transfected into cells? Product FAQ

Answer

We recommend using the Neon Transfection System or the Lipofectamine CRISPRMAX Cas9 Transfection Reagent.

Answer Id: E13076

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How does the Vitronection (VTN-N) Recombinant Human Protein, Truncated compare to wild type vitronectin? Product FAQ

Answer

The truncated form of vitronectin supports human pluriportent stem cells attachment and survival better than wild-type vitronectin. This truncated form has been optimized for use with Essential 8 Medium.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Answer Id: E13080

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Can Dopaminergic Neuron Maturation medium be used to differentiate neural stem cells? Product FAQ

Answer

We have tested neural stem cells (NSCs) isolated from fetal tissue or derived from pluripotent stem cells (PSCs), and have seen that both populations can benefit from maturation medium (DMEM/F12 + Dopaminergic Neuron Maturation Supplement) to have nicely spread homogenous neurons with reduced progenitor population. Matured neurons can be further maintained in neurobasal medium supplemented with Dopaminergic Neuron Maturation Supplement.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Answer Id: E13084

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What is the recommended reaction temperature for SuperScript IV VILO Master Mix? Product FAQ

Answer

The recommended reaction temperature for SuperScript IV VILO Master Mix is 50 degrees C. R&D has tested the stability of SSIV VILO at higher temperatures and it works very well even at 65 degrees C.

Answer Id: E13088

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What are the Leibovitz media you offer? Product FAQ

Answer

We offer the following Leibovitz media:

- Leibovitz's L-15 Medium, powder: Cat. Nos. 41300039 (10x 1L) and 41300070 (1x 10L)
- Leibovitz's L-15 Medium, no phenol red: Cat. No 21083027 (1x 500 mL)
- Leibovitz's L-15 Medium: Cat. Nos.11415064 (1x 500 mL) and 11415114 (10x 500 mL) (for sale in N. America, Latin America, and Asia-Pacific regions; Cat. No. 11415049 (for sale in Europe, Africa, and the Middle East
- Leibovitz's L-15 Medium with GlutaMAX Supplement: Cat. No. 31415029 (1x 500 mL)

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Answer Id: E13258

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Why do you offer HBSS with and without phenol red? Product FAQ

Answer

Phenol red is a pH indicator which allows you to determine the optimal atmospheric conditions for your cell line during your particular assays. We offer both versions to allow you the option to utilize this indicator in your media.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Answer Id: E13270

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What is the advantage of using Insulin-Transferrin-Selenium (ITS-G) Supplement in cultures? Product FAQ

Answer

ITS-G Supplement was designed to supplement RPMI-1640 or Earle's Minimal Essential Medium (EMEM), and will enhance the growth of various cell types at FBS concentrations < 4%.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Answer Id: E13278

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