TransAM® Flexi NFκB Kits

All of the TransAM kits will work with human samples. Some have cross-reactivity with mouse or rat. Each TransAM manual contains cross-reactivity information under the Kit Performance and Benefits section.

Each of our specific TransAM kits has a different detection limit, which is indicated in each manual. For example, our TransAM NF-kB kits can detect binding from as little as 0.5 ug nuclear extract. Using a rough estimate yield of 20 ug nuclear extract from 1 million cells from our Nuclear Extract Kit, 0.5 ug nuclear extract can be obtained from approximately 25,000 cells.

The optimal protein concentration must be empirically determined. We recommend performing a small titration experiment– 5 ug, 10 ug, 20 ug for nuclear extract, more for whole cell extract, in order to ensure that the amount used per well is within detectable levels. You want to get both the treated and control sample signals within the linear range of the assay. See the TransAM color chart.

Yes, you can increase the total volume of sample added and scale up the reaction, but the ratio of Complete Binding Buffer to Complete Lysis Buffer must remain the same. Please note that there may not be enough reagents to perform all 96-rxns if you scale up and do not exceed a total volume of 100 µl. (Complete Binding Buffer plus sample diluted in Complete Lysis Buffer)

The purpose of the reference wavelength (OD_655nm) is to correct or normalize any differences in absorbance that are not due to the analyte. Everything being equal, the reference absorbance should not change between samples. But because of factors like variability in detection equipment, wells, etc. the absorbance can vary. Subtracting the reference from the specific reading: OD_450nm - OD_655nm accounts for these variations. Often the spectrophotometer or the program automatically does this.

We recommend at a minimum running the assay in duplicate to get an idea of technical variability. If your assay has a high variability, we recommend doing triplicates. Plot the data with standard error to get a measure of the variability.

The R² value of the linear regression line should be 0.94 or higher for the standard curve. To increase this value, perform more replicates and pipet with precision.

Generally speaking, a chemiluminescent ELISA is more sensitive and offers a greater dynamic range as compared to colorimetric ELISAs.

Our TransAM family kits come with two 96-well plates and antibodies against multiple transcription factors within a family. Each antibody is provided in enough abundance to cover one whole 96-well plate, so there is flexibility to assay two or more related antibodies. Our TransAM individual kits each come with one 96-well plate and enough of one antibody to cover that plate.

Not all NF-kB sites are the same and our Flexi kits allow users to design and bind their own custom DNA oligonucleotides to the plate. This allows for the study of NF-kB dimers which bind to specific endogenous DNA sequences.

Any of the following are compatible.

• Active Motif’s Nuclear Extract Kit (Cat. No. 40010/40410)**
• Preparation of Nuclear Extract protocol in the Appendix of the TransAM manual.
• Your own nuclear extract protocol, as long as the lysis buffers do not contain SDS. Be sure to resuspend the nuclear pellet in Complete Lysis Buffer.

** Note that some TransAM kits contain Lysis Buffer AM1 while others contain Lysis Buffer AM2. If working with a TransAM kit containing Lysis Buffer AM1, use the Nuclear Extract Kit as written. If working with a TransAM kit containing Lysis Buffer AM2, prepare the Complete Lysis Buffer in the Nuclear Extract Kit using Lysis Buffer AM2 rather than the AM1 that comes in the kit. If you have already prepared your samples using the Lysis Buffer AM1 from the Nuclear Extract Kit, we recommend that you dilute these 1:3 with the Lysis Buffer AM2 that came with your TransAM assay.

The Active Motif Nuclear Extract Kit (Cat. No. 40010/40410) contains a protocol for creating a nuclear extract from tissue. You can also use your own tissue nuclear extract protocol as long as you resuspend the nuclear pellet in the Complete Lysis Buffer recommended in the TransAM kit manual. Avoid using SDS as it can interfere with the assay.

Under a phase contrast microscope intact cells should appear as a dark central region (nucleus) surrounded by a halo of less dense cytoplasm. In lysed cells, the nuclei will appear as dots surrounded by asymmetric debris. See example below:

Figure: Images under phase contrast microscope of Jurkat cells before (A) and after (B) cell lysis; published with permission from Ziwei Wang, Tsinghua University.

PBMCs are more difficult to lyse and can be resistant to lysis by the Hypotonic Buffer. Verify cell lysis efficiency using phase-contract microscopy. If cells are not adequately lysed, use a dounce homogenizer with a small clearance pestle (0.025–0.076 mm) to homogenize the sample or draw the sample through a 28-30 gauge needle several times for mechanical shearing. Active Motif’s Dounce Homogenizers for 1 mL capacity (Cat. No. 40401) and 15 mL capacity (Cat. No. 40415) both come with a small and large clearance pestle.

Primary cells can be challenging because they are typically fragile and can easily lyse with just scraping. Also, the number of cells is often limited. Be sure to monitor cell lysis under the microscope to maximize the extraction yield.

It’s best to aliquot the extracts so that freeze-thaw cycles are not required. However, if you must thaw, we do not recommend freeze-thawing more than 2 times. Multiple freeze-thaws degrade proteins and destroy protein activity. Thaw the extracts on ice using pipetting to mix. Do you not vortex protein samples vigorously.

We recommend using fresh cells or tissue, as the freezing process can disrupt the cytoplasmic and nuclear compartments, making it difficult to get pure fractions. However, if necessary, snap freeze tissues at -80°C. Cryopreserve cells before freezing at -80°C.

To enhance the solubility of the pellet:

• Pipet up and down a few times with a large bore pipette tip.
• Use a dounce homogenizer.
• Vortex on high for 10 seconds.
• Increase the 30-minute incubation on the rocking platform to an hour.

If needed add 5-10 ul of Complete Lysis Buffer. Be sure not to overdilute the nuclear sample. Note that certain cell types are more likely to produce a viscous nuclear pellet and yet still give good protein yields, even if it doesn’t completely disappear. Do not add detergent as it could interfere with the TransAM assay.

It depends. With a whole-cell extract, the transcription factor of interest will be diluted into a larger volume than in a nuclear extract. So, more protein (2x or more) will be required. Also, if the transcription factor is regulated by nuclear-cytoplasmic localization, using whole-cell extract could affect the functional aspect of the assay.

It is not recommended for several reasons. The TransAM kit is designed to measure protein-DNA binding, which exclusively occurs in the nucleus. Any signal with the cytoplasmic extract may not be physiologically relevant. Also, the cytoplasmic extract is going to be much more dilute as compared to nuclear extract and will contain detergents unlike the nuclear fraction that that may interfere with the assay.

We recommend using a Bradford Assay to quantify the protein rather than BCA. A BCA assay requires a higher dilution factor to eliminate confounding interfering agents and can limit the detection of protein in all but very concentrated samples. If using the Nuclear Extract Kit (Cat. No. 40010/40410), we recommend a dilution factor of 1:50 in water. There is a protocol for creating a BSA standard curve in the Appendix of the Nuclear Extract Kit manual.

We suggest isolating nuclear extracts from samples of approximately 8.8 x10⁶ cells or 45 mg tissue. A tissue conversion factor for general estimation is 2x10⁵ cells/mg.

This will depend on your cell or tissue type, but in general you should expect 150-250 µg at ~3-5 mg/ml from 8.8 x 10⁶ cells.

SDS or sodium azide in your cell lysis buffer will inhibit the binding and can render your sample incompatible.