MID1 Antibody Protocol: Applications & Usage Guide
MID1 Antibody Protocol: Applications & Usage Guide
MID1 Antibody is a research-grade immunological reagent designed for the specific detection of the MID1 protein, a RING-domain E3 ubiquitin ligase implicated in X-linked Opitz syndrome and microtubule-associated regulatory pathways.
- Product Name
- MID1 Antibody
- SKU
- Ab351010
- Formulation
- PBS only (no carrier protein, no preservative)
- Target Protein
- MID1 (Midline-1), an E3 ubiquitin ligase associated with X-linked Opitz syndrome
- Recommended Storage
- Store at -20°C; avoid repeated freeze-thaw cycles
- Reference Source
- Absea #OC-2536
Background: MID1 Protein and Its Biological Role
MID1 (Midline-1), also known as TRIM18, is a member of the tripartite motif (TRIM) protein family. It contains an N-terminal RING domain with E3 ubiquitin ligase activity, two B-box zinc-finger motifs, and a coiled-coil domain. MID1 associates with microtubules and plays a critical role in regulating the degradation of the catalytic subunit of protein phosphatase 2A (PP2Ac) via the ubiquitin-proteasome pathway.
Loss-of-function mutations in the MID1 gene are causally linked to X-linked Opitz G/BBB syndrome, a congenital disorder characterized by midline developmental defects including hypertelorism, cleft palate, and hypospadias. MID1 has also been implicated in mTOR signaling, Huntingtin protein regulation, and cancer biology, making it a target of broad research interest.
Sample Preparation and Recommended Dilutions
Proper sample preparation is essential for optimal MID1 Antibody performance. Follow the guidelines below for each application:
- Western Blot (WB): Lyse cells or tissue in RIPA buffer supplemented with protease inhibitors. Separate 20–40 µg of total protein by SDS-PAGE and transfer to PVDF or nitrocellulose membrane. Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature. Incubate with MID1 Antibody at the manufacturer's recommended dilution (see product datasheet; specific dilution not stated in current source material — consult Absea #OC-2536 datasheet) overnight at 4°C.
- Immunohistochemistry (IHC): Use formalin-fixed, paraffin-embedded (FFPE) sections. Perform antigen retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). Block endogenous peroxidase activity before primary antibody incubation. Apply MID1 Antibody at the recommended dilution and incubate overnight at 4°C.
- Immunofluorescence (IF): Fix cells with 4% paraformaldehyde and permeabilize with 0.1–0.3% Triton X-100. Block with 5% normal serum for 1 hour. Incubate with MID1 Antibody at the recommended dilution for 1 hour at room temperature or overnight at 4°C, followed by appropriate fluorescently labeled secondary antibody.
Note: Specific working dilutions are provided on the product datasheet (Absea #OC-2536). Always perform a pilot titration experiment to determine the optimal dilution for your specific experimental system.
Storage, Handling, and Formulation Notes
MID1 Antibody (Ab351010) is formulated in PBS only, without carrier proteins such as BSA or glycerol, and without preservatives such as sodium azide. This formulation is particularly suitable for applications requiring conjugation or downstream functional assays where carrier proteins or preservatives may interfere.
Storage Recommendations:
- Store at -20°C for long-term storage.
- For short-term use (up to 1 month), the antibody may be stored at 4°C.
- Avoid repeated freeze-thaw cycles, which can lead to antibody aggregation and loss of activity. Aliquot the antibody upon first thaw if repeated access is anticipated.
- Do not store in frost-free freezers, which undergo temperature cycling.
Handling Tips:
- Briefly centrifuge the vial before opening to collect contents at the bottom.
- Keep on ice during use to maintain antibody integrity.
- Use low-binding microcentrifuge tubes to minimize adsorptive losses when working with diluted antibody solutions.
Troubleshooting Common Issues
The following troubleshooting guide addresses common challenges encountered when using MID1 Antibody:
| Problem | Possible Cause | Suggested Solution |
|---|---|---|
| No signal / weak signal | Antibody concentration too low; suboptimal antigen retrieval; target protein not expressed in sample | Increase antibody concentration; optimize antigen retrieval method and duration; confirm MID1 expression in your cell/tissue type using literature; use positive control lysate |
| High background | Insufficient blocking; antibody concentration too high; non-specific secondary antibody binding | Increase blocking time or use alternative blocking agent (e.g., switch from milk to BSA); reduce primary antibody concentration; titrate secondary antibody |
| Multiple non-specific bands (WB) | Protein degradation; cross-reactivity | Add fresh protease inhibitors during lysis; verify sample integrity by loading quality control; consult the product datasheet for expected molecular weight of MID1 |
| Inconsistent results between experiments | Freeze-thaw damage; pipetting variability | Aliquot antibody to avoid repeated freeze-thaw; use calibrated pipettes; standardize protein loading |
Frequently asked questions
What applications is MID1 Antibody (Ab351010) validated for?
The specific validated applications for Ab351010 should be confirmed on the product datasheet (Absea #OC-2536). MID1 antibodies are commonly used in Western blot (WB), immunohistochemistry (IHC), and immunofluorescence (IF). Please refer to the official Aladdin Scientific product page or contact technical support for the confirmed application list.
Why is MID1 Antibody formulated in PBS only, and does this affect my experiment?
The PBS-only formulation (without BSA, glycerol, or preservatives) makes MID1 Antibody (Ab351010) highly suitable for conjugation reactions (e.g., fluorescent dye or enzyme labeling) and functional assays where carrier proteins or sodium azide could interfere with downstream applications. However, this formulation may require more careful handling and storage to preserve antibody stability — aliquoting and avoiding freeze-thaw cycles are strongly recommended.
What positive control should I use when testing MID1 Antibody?
For Western blot, cell lines or tissues known to express MID1 protein are suitable positive controls. MID1 is broadly expressed, with reported expression in kidney, brain, and various cancer cell lines. Consult published literature to identify an appropriate positive control for your specific application. A recombinant MID1 protein control may also be used if available.
How should I aliquot MID1 Antibody to prevent freeze-thaw degradation?
Upon first thaw, briefly centrifuge the stock vial, then divide the antibody into single-use volumes using low-binding microcentrifuge tubes. Label each aliquot with the date and antibody concentration. Store aliquots at -20°C and thaw only what is needed for each experiment to avoid repeated freeze-thaw cycles that may reduce antibody activity.
References
- Absea Biotechnology. Product datasheet for MID1 Antibody, catalog #OC-2536. Absea Biotechnology Ltd.
- Quaderi NA, et al. Opitz G/BBB syndrome, a defect of midline development, is due to mutations in a new RING finger gene on Xp22. Nature Genetics. 1997;17(3):285-291.
- Trockenbacher A, et al. MID1, mutated in Opitz syndrome, encodes an ubiquitin ligase that targets phosphatase 2A for degradation. Nature Genetics. 2001;29(3):287-294.
Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.