Recombinant Prothrombin Antibody Protocol | Detection & Usage

Recombinant Prothrombin Antibody is a high-specificity reagent designed for the detection of prothrombin (coagulation factor II) in research applications including Western blot, immunofluorescence, and ELISA.

Product Name

Recombinant Prothrombin Antibody

SKU

Ab345382

Lot Concentration

1.25 mg/ml

Supplier Reference

Abcam23453; Purchase Order P240013083; Supplier Lot F029207

Format

Recombinant antibody, liquid

Target

Prothrombin (Coagulation Factor II, F2)

Materials and Reagents

Before beginning the protocol, ensure the following materials are available:

• Recombinant Prothrombin Antibody (Ab345382) at 1.25 mg/ml stock concentration
• Appropriate blocking buffer (e.g., 5% non-fat dry milk or 3% BSA in TBST)
• Primary antibody diluent (PBS or TBST with 1–3% BSA)
• Secondary antibody compatible with the host species of this recombinant antibody
• Wash buffer: TBST (Tris-buffered saline with 0.1% Tween-20)
• Detection reagents appropriate for the chosen application (ECL substrate for WB; HRP-conjugate or fluorescent secondary for ELISA/IF)
• Standard laboratory equipment: microcentrifuge, pipettes, membrane or plate as required

Note: Always verify the lot number (F029207) and concentration (1.25 mg/ml) printed on the vial label before use.

Antibody Preparation and Dilution

Proper preparation of the antibody is critical for reproducible results. Follow the steps below:

1. Thaw: If stored frozen, thaw the antibody vial on ice or at 2–8 °C. Avoid repeated freeze-thaw cycles to preserve activity.
2. Centrifuge: Briefly centrifuge the vial (300 × g, 30 seconds) to collect any liquid from the cap.
3. Prepare working dilution: Dilute the antibody from the 1.25 mg/ml stock into the appropriate diluent. Recommended starting dilutions are:
   • Western Blot (WB): 1:500–1:2000
   • ELISA: 1:1000–1:5000
   • Immunofluorescence (IF): 1:100–1:500
4. Mix gently: Do not vortex. Invert or gently pipette to mix.

Optimal dilution should be determined empirically for each experimental system and sample type.

Detection Protocol (Western Blot Example)

The following steps outline a standard Western blot protocol using the Recombinant Prothrombin Antibody:

1. Sample preparation: Prepare protein lysates from relevant cell lines or tissue samples. Prothrombin is predominantly expressed in the liver and present in plasma.
2. Electrophoresis: Resolve 20–40 µg total protein by SDS-PAGE on an appropriate percentage gel (recommended: 8–10% for prothrombin, ~72 kDa).
3. Transfer: Transfer proteins to PVDF or nitrocellulose membrane using standard wet or semi-dry transfer conditions.
4. Blocking: Block membrane for 1 hour at room temperature with blocking buffer.
5. Primary antibody incubation: Incubate membrane with diluted Recombinant Prothrombin Antibody (Ab345382) overnight at 4 °C with gentle rocking.
6. Washing: Wash 3 × 10 minutes with TBST.
7. Secondary antibody: Incubate with appropriate HRP-conjugated secondary antibody for 1 hour at room temperature.
8. Washing: Wash 3 × 10 minutes with TBST.
9. Detection: Apply ECL substrate and image using a chemiluminescence imaging system or X-ray film.

Expected band size for prothrombin: approximately 72 kDa (precursor form). Post-translational modifications may alter apparent molecular weight.

Storage and Handling

Correct storage is essential to maintain antibody integrity and activity:

• Short-term (up to 1 month): Store at 2–8 °C.
• Long-term: Aliquot and store at −20 °C or −80 °C. Avoid repeated freeze-thaw cycles.
• Avoid: Exposure to light, repeated freeze-thawing, and temperatures above 25 °C during handling.
• Lot-specific information: This protocol applies to lot F029207 at a concentration of 1.25 mg/ml (sourced via Abcam23453, PO P240013083). Always verify current lot information on the Certificate of Analysis (CoA).

If the antibody has been stored at −20 °C, allow complete thawing on ice before use. Do not use a water bath above 37 °C for thawing.

Troubleshooting

If unexpected results are obtained, consider the following troubleshooting steps:

Problem	Possible Cause	Suggested Action
No signal detected	Antibody concentration too low; insufficient antigen	Increase antibody concentration; verify target expression in sample; check secondary antibody compatibility
High background	Antibody concentration too high; insufficient blocking or washing	Increase dilution; extend blocking time; increase number or duration of washes
Non-specific bands	Cross-reactivity; sample degradation	Add protease inhibitors to lysis buffer; optimize dilution; verify sample integrity
Weak or faint signal	Over-dilution; degraded antibody from improper storage	Reduce dilution factor; confirm antibody was stored correctly; check lot expiry

Always include a positive control (e.g., human liver lysate or plasma sample) and a negative control (primary antibody omitted) in each experiment.

Frequently asked questions

What is the stock concentration of Recombinant Prothrombin Antibody (Ab345382)?

The stock concentration for lot F029207 is 1.25 mg/ml. Always verify the concentration on the vial label and the Certificate of Analysis before preparing working dilutions.

How should I store the Recombinant Prothrombin Antibody to maintain its activity?

For short-term use (up to one month), store at 2–8 °C. For long-term storage, aliquot the antibody and store at −20 °C or −80 °C. Avoid repeated freeze-thaw cycles, exposure to light, and temperatures above 25 °C during handling.

Which applications is the Recombinant Prothrombin Antibody suitable for?

This antibody is suitable for research applications including Western blot, ELISA, and immunofluorescence. Optimal working dilutions should be determined empirically for each application and sample type.

What positive control should I use with this antibody?

Human liver lysate or human plasma is recommended as a positive control, as prothrombin (coagulation factor II) is predominantly synthesized in the liver and is abundant in plasma. Including a negative control (primary antibody omitted) is also strongly advised.

What is the expected molecular weight of prothrombin on a Western blot?

The expected band size for prothrombin (coagulation factor II) is approximately 72 kDa for the precursor form. Post-translational modifications such as glycosylation may alter the apparent molecular weight on SDS-PAGE.

References

1. Abcam. Recombinant Prothrombin Antibody datasheet. Source reference: Abcam23453, Supplier Lot F029207.
2. Mann KG. Prothrombin. Methods Enzymol. 1976;45:123-156. [Textbook-level reference for prothrombin biochemistry]

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.