Image 1: FACL4, also known as acyl-CoA synthetase 4 (ACSL4), target protein structure.

FACL4 Introduction

Protein Function

• FACL4 can catalyze the conversion of long-chain fatty acids into their active form, acyl-CoA, which is involved in both cellular lipid synthesis and degradation through β-oxidation.
• FACL4 preferentially activates arachidonic acid esters and eicosapentaenoic acid esters as substrates.
• FACL4 mediates the production of 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE), which are crucial predictive factors for iron death susceptibility.
• FACL4 can regulate the secretion of prostaglandin E2.
• Defects in FACL4 can lead to X-linked intellectual disability 63 (MRX63), which is associated with intellectual disability, midfacial hypoplasia, and elliptocytosis in Alport syndrome.

Protein Expression

• FACL4 is highly expressed in tissues with active steroid synthesis, such as the adrenal glands, ovaries, and testes.
• FACL4 is expressed at lower levels in breast, gastrointestinal, and cardiac tissues.
• Compared to normal tissues, the expression of FACL4 is upregulated in some tumor tissues, such as colon adenocarcinoma, hepatocellular carcinoma, and breast cancer.

Protein Localization

• Outer mitochondrial membrane, peroxisomal membrane, microsomal membrane, endoplasmic reticulum membrane, and cell membrane.

Image 2: ICC experimental result of FACL4 protein, Anti-FACL4 antibody [EPR17587-42] (ab205199). Green: FACL4, Red: Tubulin, Blue: DAPI.

Isoforms & Post-translation modifications

• Human (O60488):Isoforms O60488-1~ O60488-2: 74.4-79.2 kDa (predicted)
• Mouse (Q9QUJ7):Isoforms Q9QUJ7-1~ Q9QUJ7-2: 74.3-79.1 kDa (predicted)
• Rat (O35547):Isoforms O35547-1~ O35547-2: 74.3-79.1 kDa (predicted)
• Phosphorylation modification is present.

WB experiment tips

Precautions

• FACL4 is expressed differently in different tissues. If you are unsure about the expression of the target protein in the sample to be tested, please select tissues with vigorous steroid synthesis, such as adrenal glands and ovarian tissues, or cell lines validated by the antibody product instructions, such as HepG2 and HeLa cells, as positive controls for the experiment.
• If brain tissue is chosen for WB experiment, it is important to note that there are significant differences in the expression of FACL4 in different brain regions. To avoid no signal or weak signal in the experimental results, it is recommended to prioritize the detection of FACL4 in components with higher content, such as the hippocampus and cerebellum, or select other samples with high expression of FACL4 as positive controls, while increasing the concentration of the primary antibody.
• It is recommended not to strip the membrane, or to retain the range of 50-250 kDa, to prevent loss of protein signal.
• Due to the presence of isoforms, FACL4 may exhibit double band phenomenon in WB detection.

Positive controls

• Human: HepG2, HeLa whole cell lysates
• Mouse: Brain tissue lysates
• Rat: Brain tissue, kidney tissue lysates

Example of results

Figure 3: WB experimental results of FACL4 protein, Anti-FACL4 antibody [EPR8640] (ab155282).

Lane 1: Rat brain tissue lysate
Lane 2: Rat heart tissue lysate
Lane 3: Rat kidney tissue lysate
Lane 4: Rat spleen tissue lysate

Primary antibody dilution ratio: 1/10000
Predicted band size: 79 kDa
Observed band size: 75 kDa

Figure 4: WB experiment result image of FACL4 protein, using Anti-FACL4 antibody [EPR17587-42] (ab205199).

Lane 1: HeLa whole cell lysate
Lane 2: HepG2 whole cell lysate

Loading amount: 20 μg
Predicted band size: 79 kDa
Observed band size: 79 kDa
Exposure time: 30 s

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample preparation:

1. Add a complex protease inhibitor to avoid degradation of the target protein.
2. Select a suitable lysis buffer to enrich more target proteins.
3. Keep the sample on ice throughout the sample preparation process.
4. Determine the protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. Load at least 20μg total protein for electrophoresis.
2. It is recommended to use positive and negative controls.

Transfer:

1. After activation of the PVDF membrane, thoroughly wash it to completely remove residual methanol on the membrane.
2. It is recommended to use Ponceau S staining after transfer to confirm the success of the transfer (if fluorescence labeling detection is chosen, make sure Ponceau S is completely washed off).

Blocking:

1. There is no blocking solution that is suitable for all systems, so please choose the appropriate blocking solution.

Reference

1. Golej D.L, Askari B, Kramer F, et al. Long-chain acyl-CoA synthetase 4 modulates prostaglandin E(2) release from human arterial smooth muscle cells. J Lipid Res. 2011;52(4):782-93.
2. Ohkuni A, Ohno Y, Kihara A. Identification of acyl-CoA synthetases involved in the mammalian sphingosine 1-phosphate metabolic pathway. Biochem Biophys Res Commun. 2013 ;442(3-4):195-201.
3. Hua Yuan, Xuemei Li, Xiuying Zhang, et al. Identification of ACSL4 as a biomarker and contributor of ferroptosis. Biochem Biophys Res Commun. 2016 ;478(3):1338-43.
4. Meloni I, Muscettola M, Raynaud M, et al. FACL4, encoding fatty acid-CoA ligase 4, is mutated in nonspecific X-linked mental retardation. Nat Genet. 2002 ;30(4):436-40.
5. Piccini M, Vitelli F, Bruttini M, et al. FACL4, a new gene encoding long-chain acyl-CoA synthetase 4, is deleted in a family with Alport syndrome, elliptocytosis, and mental retardation. Genomics. 1998 ;47(3):350-8.