Figure 1: GLUT1 protein structure.

Introduction to GLUT1

Protein Function

• GLUT1 is the facilitative glucose transporter responsible for constitutive or basal glucose uptake.
• It has a broad substrate specificity and can transport many aldoses, including pentoses and hexoses.
• The brain's most crucial energy carrier is present at the blood-brain barrier and ensures the energy-independent, facilitative transport of glucose into the brain.

Protein Expression

• Detected in erythrocytes (at protein level).
• Expressed at variable levels in many human tissues.

Protein Localization

• Localizes primarily at the cell surface.
• Identified by mass spectrometry in melanosome fractions from stage I to stage IV.

Figure 2: GLUT1 ICC image, Anti-Glucose Transporter GLUT1 antibody [EPR3915] (ab115730).

Isoforms & Post-translation Modifications

Human (P11166): 54 kDa (predicted)
Mouse (P17809): 54 kDa (predicted)
Rat (P11167): 54 kDa (predicted)
Post-translation modifications include acetylation, phosphorylation, and glycosylation.

WB Experimental Tips

Precautions

• GLUT1 undergoes glycosylation modifications, and its actual detected molecular weight is typically around 40-60 kDa (predicted molecular weight: 54 kDa), which may result in multiple banding patterns.
• We do not recommend boiling samples after lysis. GLUT1 is a multi-pass membrane protein, and high-temperature boiling can lead to protein aggregation, preventing effective entry into the gel and causing band broadening, fuzziness, or trailing.
• Samples should be dissolved in standard SDS Laemmli buffer.

Positive Controls

• HepG2 cell lysate, HT-29 cell lysate

Example Results

Figure 3: Anti-Glucose Transporter GLUT1 antibody [EPR3915] (ab115730).

Lane 1: 10 µg Jurkat whole cell lysate.
Lane 2: 10 µg mouse brain lysate.
Lane 3: 10 µg human fetal brain lysate.
Lane 4: 10 µg 3T3L1 whole cell lysate.
Lane 5: 10 µg human fetal liver lysate.
Lane 6: 10 µg HepG2 whole cell lysate.

Figure 4: Anti-Glucose Transporter GLUT1 antibody [EPR3915] (ab115730).

Lane 1:20 µg HT-29 whole cell lysates unboiled with 5% NFDM/TBST.
Lane 2:20 µg HT-29whole cell lysates unboiled with 5% NFDM/TBST.
Lane 3:20 µg 3T3-L1 whole cell lysates unboiled with 5% NFDM/TBST.
Lane 4:20 µg 3T3-L1 whole cell lysates boiled with 5% NFDM/TBST.

Predicted band size: 54 kDa.
Observed band size: 40-60 kDa.
Other: We recommend not boiling the sample after lysis to get desired WB bands.

Key control points

In the experiment, alongside routine issues, key control points require special attention.

Sample preparation:

1. Add a protease inhibitor cocktail to prevent degradation of target proteins.
2. Keep samples on ice throughout the entire sample preparation process.
3. We recommend not boiling the samples after lysis
4. Determine the protein concentration of the samples using Bradford analysis, Lowry analysis, or BCA analysis.

Transfer:

1. We recommend staining the membrane with Ponceau S after the transfer to confirm its success.

References

1. Mike Mueckler, Carol Makepeace. Transmembrane segment 6 of the Glut1 glucose transporter is an outer helix and contains amino acid side chains essential for transport activity. J Biol Chem. 2008 Apr 25;283(17):11550-5. doi: 10.1074/jbc.M708896200. Epub 2008 Feb 1.
2. Khyati Kapoor, Janet S Finer-Moore, Bjørn P Pedersen, etc. Mechanism of inhibition of human glucose transporter GLUT1 is conserved between cytochalasin B and phenylalanine amides. Proc Natl Acad Sci U S A. 2016 Apr 26;113(17):4711-6. doi: 10.1073/pnas.1603735113. Epub 2016 Apr 12.
3. Mike Mueckler, Carol Makepeace. Model of the exofacial substrate-binding site and helical folding of the human Glut1 glucose transporter based on scanning mutagenesis. Biochemistry. 2009 Jun 30;48(25)10.1021/bi900521n.
4. J Klepper, D Wang, J Fischbarg, J C Vera, I T Jarjour, K R O'Driscoll, D C De Vivo. Defective glucose transport across brain tissue barriers: a newly recognized neurological syndrome. Neurochem Res. 1999 Apr;24(4) 10.1023/a:1022544131826.