Overview of NFAT5 and Antibody Specifications

NFAT5, also known as TonEBP (Tonicity-responsive Enhancer Binding Protein), is a transcription factor belonging to the Rel homology domain superfamily. Unlike other NFAT family members, NFAT5 is not regulated by calcineurin-dependent calcium signaling; instead, it is activated by hyperosmotic stress and plays a central role in cellular osmoprotection by inducing the expression of genes encoding osmolyte transporters and biosynthetic enzymes.

The NFAT5 Mouse mAb (Ab351130) is a monoclonal antibody produced in mouse, targeting NFAT5 protein. It is supplied in a PBS-only formulation (absea reference KC-641), making it compatible with downstream conjugation or direct dilution into assay buffers. The monoclonal nature of this antibody ensures lot-to-lot consistency and high specificity for the NFAT5 epitope.

Materials and Equipment Required

• NFAT5 Mouse mAb (Ab351130), PBS formulation
• Appropriate cell lysate or tissue homogenate containing NFAT5 protein
• Standard immunoassay buffers: TBST or PBST (0.1% Tween-20)
• Blocking reagent: 5% non-fat dry milk or BSA in TBST
• Secondary antibody: anti-mouse IgG conjugated to HRP or fluorescent dye
• Detection substrate appropriate for secondary antibody label
• Microcentrifuge, SDS-PAGE apparatus, PVDF or nitrocellulose membrane (for western blot)
• Microscope or plate reader, as appropriate for application
• Protease inhibitor cocktail for lysis buffer preparation

Note: Specific dilutions and buffer conditions should be empirically optimized for each application and sample type.

Step-by-Step Protocol

1. Antibody Preparation

Thaw the NFAT5 Mouse mAb (Ab351130) on ice. Gently mix by pipetting; do not vortex. Centrifuge briefly at low speed to collect contents. Prepare working dilutions in the appropriate assay buffer immediately before use. Because the antibody is supplied in PBS only (without stabilizing additives such as glycerol or BSA), prepare aliquots before the first use to minimize freeze-thaw cycles.

2. Sample Preparation

Prepare cell or tissue lysates using a lysis buffer supplemented with protease inhibitors. Quantify total protein by BCA or Bradford assay. For western blot, denature samples in SDS loading buffer at 95 °C for 5 minutes. Resolve proteins by SDS-PAGE and transfer to PVDF or nitrocellulose membrane.

3. Blocking

Block the membrane or slide with 5% non-fat dry milk or 3–5% BSA in TBST for 1 hour at room temperature with gentle agitation to minimize non-specific binding.

4. Primary Antibody Incubation

Dilute the NFAT5 Mouse mAb to the recommended working concentration in blocking buffer. Incubate with the membrane or sample overnight at 4 °C with gentle agitation. Wash three times with TBST, 5 minutes each.

5. Secondary Antibody Incubation and Detection

Incubate with an appropriate anti-mouse IgG secondary antibody at room temperature for 1 hour. Wash three times with TBST. Apply detection substrate and image according to the secondary antibody manufacturer's instructions.

Storage, Handling and Troubleshooting

Storage

Store the NFAT5 Mouse mAb at –20 °C for long-term storage. For short-term use (up to 1 month), the antibody may be stored at 4 °C. Because the PBS-only formulation lacks cryoprotectants such as glycerol, it is especially important to aliquot the stock into single-use volumes prior to freezing to prevent activity loss from repeated freeze-thaw cycles.

Handling Precautions

Always handle antibodies on ice. Avoid prolonged exposure to room temperature. Do not use antibody solutions that appear turbid or show visible precipitation without first centrifuging and re-testing for activity.

Troubleshooting