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Glut1 Antibody - BSA Free

Catalog # NB110-39113 | Novus Biologicals a Bio-Techne Brand

Key Product Details

Validated by

Biological Validation

Species Reactivity

Human, Mouse, Rat, Rabbit

Applications

Chromatin Immunoprecipitation, Chromatin Immunoprecipitation (ChIP), Flow (Intracellular), Flow Cytometry, Immunocytochemistry/ Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Frozen, Immunohistochemistry-Paraffin, In vitro assay, Western Blot

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free

Concentration

1 mg/ml

Product Summary for Glut1 Antibody - BSA Free

Immunogen

This Glut1 antibody is made against a synthetic peptide made to an N-terminal region of the human GLUT1 protein (between residues 1-100). [Swiss-Prot# P11166]. The immunogen is cytosolic.

Predicted Species

Bovine (93%), Primate (100%). Backed by our 100% Guarantee.

Reactivity Notes

Rabbit reactivity reported in scientific literature (PMID: 29456650). 100% sequence identity with primate, 93% sequence identity with bovine.

Localization

Cell membrane, cytoplasm (near membranes), melanosome

Marker

Plasma Membrane Marker

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

54.1 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for Glut1 Antibody - BSA Free

Western Blot: Glut1 Antibody [NB110-39113] - PrP-mediated increase in IC iron downregulates glucose transporters in the brain, neuroretina, and the liver: A similar evaluation of retinal lysates shows upregulation of Glut1 in PrP-/- relative to Tg40 PrP samples (lanes 1 & 3). Iron overloading downregulates Glut1 in Tg40 PrP samples relative to untreated controls, but has minimal effect on similarly treated PrP-/- samples (lanes 2 & 4). Image collected and cropped by CiteAb from the following publication (https://www.nature.com/articles/s41598-018-24786-1) licensed under a CC-BY license.
Western Blot: Glut1 Antibody [NB110-39113] - Western blot of GLUT1 on mouse kidney membrane protein (lane A) and rat kidney membrane protein (lane B).
Immunohistochemistry-Paraffin: Glut1 Antibody [NB110-39113] - Immunohistochemical analysis of FFPE tissue section of human placenta using 1:200 dilution of Glut1 antibody. The staining was developed using HRP-DAB detection method and the sections were further counterstained with hematoxylin. This antibody generated a specific strong membrane cytoplasmic staining of Glut1 primarily in the syncytiotrophoblast layers of various villi and in the red blood cells (RBCs). Cytotrophoblasts showed a very weak expression of this protein.

Applications for Glut1 Antibody - BSA Free

Application
Recommended Usage

Chromatin Immunoprecipitation

reported in scientific literature

Flow (Intracellular)

1 ug/ml

Flow Cytometry

1 ug/ml. Use reported by customer review

Immunocytochemistry/ Immunofluorescence

1:1000

Immunohistochemistry

1:200

Immunohistochemistry-Frozen

1:200. Use reported in scientific literature

Immunohistochemistry-Paraffin

1:200

In vitro assay

reported in scientific literature (Trachsel V et al)

Western Blot

1:500
Application Notes
In WB a band is seen at ~55 kDa on kidney membrane preps representing GLUT1 protein. Depending on the tissue and any post-translational modifications, this protein can run anywhere between 40-60 kDa.
Please Note: Optimal dilutions of this antibody should be experimentally determined.

Reviewed Applications

Read 7 reviews rated 4.9 using NB110-39113 in the following applications:

Published Applications

Read 60 publications using NB110-39113 in the following applications:

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Aliquot and store at -20C or -80C. Avoid freeze-thaw cycles.

Background: Glut1

Glucose transporter 1 (GLUT1) or solute carrier family 2 (SLC2A1) is a member of the GLUT family of monosaccharides and polyols transporters. GLUT proteins transport glucose across cellular membranes through facilitative mechanisms and play a key role in glucose homeostasis (1). Fourteen GLUT proteins have been identified in the human, which are encoded by SLC2A genes 1-14 and are broadly expressed in many cell types and tissues. GLUT family members differ in sequence homology, substrate specificity and expression patterns. Based on sequence homology, GLUT family members are classified into Class I (GLUT1, 2, 3, 4, and GLUT14), Class II (GLUT5, 7, 9, and 11), and Class III (GLUT6, 8, 10, 12 and 13) (1). Structurally, GLUT transporters are integral membrane glycoproteins consisting of 12 membrane spanning helical domains, a single N-linked glycosylation site, and having cytoplasmic facing carboxy and amino terminal domains (2).

GLUT1 (Human glycosylated form theoretical molecular weight 55kDa) functions primarily as a glucose transporter but can transport other substrates including mannose, galactose and glucosamine across the membrane (3). Like other GLUT family members, GLUT1 is broadly expressed, nevertheless it is the predominant glucose transporter expressed in red blood cells and brain endothelial cells (1). SLC2A1 mutations underscore the autosomal dominant disorder GLUT1 deficiency syndrome (GLUTI-DS) which is characterized by low glucose levels in the brain or hypoglycorrhachia due to insufficient glucose transport across the blood brain barrier (2, 4, 5). Phenotypically, GLUT1-DS is characterized by early onset seizures, neurologic developmental delay, microcephaly, and ataxia (4). GLUT1 is highly expressed in the endothelium of cutaneous vascular lesions and serves as a marker for the diagnosis of juvenile or infantile hemangiomas (6).

References

1. Augustin, R. (2010). The protein family of glucose transport facilitators: It's not only about glucose after all. IUBMB Life. https://doi.org/10.1002/iub.315

2. Mueckler, M., & Thorens, B. (2013). The SLC2 (GLUT) family of membrane transporters. Molecular Aspects of Medicine. https://doi.org/10.1016/j.mam.2012.07.001

3. Stein, W. D., & Litman, T. (2015). Carrier-Mediated Transport. In Channels, Carriers, and Pumps. https://doi.org/10.1016/b978-0-12-416579-3.00004-6

4. Pearson, T. S., Akman, C., Hinton, V. J., Engelstad, K., & De Vivo, D. C. (2013). Phenotypic spectrum of glucose transporter type 1 deficiency syndrome (Glut1 DS). Current Neurology and Neuroscience Reports. https://doi.org/10.1007/s11910-013-0342-7

5. Messana, T., Russo, A., Vergaro, R., Boni, A., Santucci, M., & Pini, A. (2018). Glucose transporter type 1 deficiency syndrome: Developmental delay and early-onset ataxia in a novel mutation of the SLC2A1 gene. Journal of Pediatric Neurosciences. https://doi.org/10.4103/JPN.JPN_169_17

6. van Vugt, L. J., van der Vleuten, C. J. M., Flucke, U., & Blokx, W. A. M. (2017). The utility of GLUT1 as a diagnostic marker in cutaneous vascular anomalies: A review of literature and recommendations for daily practice. Pathology Research and Practice. https://doi.org/10.1016/j.prp.2017.04.023

Long Name

Glucose Transporter Type 1

Alternate Names

DYT17, DYT18, DYT9, EIG12, GLUT1DS, SLC2A1

Gene Symbol

SLC2A1

Product Documents for Glut1 Antibody - BSA Free

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot number in the search box below.

Product Specific Notices for Glut1 Antibody - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

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Protocols

View specific protocols for Glut1 Antibody - BSA Free (NB110-39113):

Protocol for Flow Cytometry Intracellular Staining
Sample Preparation.
1. Grow cells to 60-85% confluency. Flow cytometry requires between 2 x 105 and 1 x 106 cells for optimal performance.
2. If cells are adherent, harvest gently by washing once with staining buffer and then scraping. Avoid using trypsin as this can disrupt certain epitopes of interest. If enzymatic harvest is required, use Accutase, Collagenase, or TrypLE Express for a less damaging option.
3. Reserve 100 uL for counting, then transfer cell volume into a 50 mL conical tube and centrifuge for 8 minutes at 400 RCF.
a. Count cells using a hemocytometer and a 1:1 trypan blue exclusion stain to determine cell viability before starting the flow protocol. If cells appear blue, do not proceed.
4. Re-suspend cells to a concentration of 1 x 106 cells/mL in staining buffer (NBP2-26247).
5. Aliquot out 100 uL samples in accordance with your experimental samples.

Tip: When cell surface and intracellular staining are required in the same sample, it is advisable that the cell surface staining be performed first since the fixation and permeabilization steps might reduce the availability of surface antigens.

Intracellular Staining.
Tip: When performing intracellular staining, it is important to use appropriate fixation and permeabilization reagents based upon the target and its subcellular location. Generally, our Intracellular Flow Assay Kit (NBP2-29450) is a good place to start as it contains an optimized combination of reagents for intracellular staining as well as an inhibitor of intracellular protein transport (necessary if staining secreted proteins). Certain targets may require more gentle or transient permeabilization protocols such as the commonly employed methanol or saponin-based methods.
Protocol for Cytoplasmic Targets:
1. Fix the cells by adding 100 uL fixation solution (such as 4% PFA) to each sample for 10-15 minutes.
2. Permeabilize cells by adding 100 uL of a permeabilization buffer to every 1 x 106 cells present in the sample. Mix well and incubate at room temperature for 15 minutes.
a. For cytoplasmic targets, use a gentle permeabilization solution such as 1X PBS + 0.5% Saponin or 1X PBS + 0.5% Tween-20.
b. To maintain the permeabilized state throughout your experiment, use staining buffer + 0.1% of the permeabilization reagent (i.e. 0.1% Tween-20 or 0.1% Saponin).
3. Following the 15 minute incubation, add 2 mL of the staining buffer + 0.1% permeabilizer to each sample.
4. Centrifuge for 1 minute at 400 RCF.
5. Discard supernatant and re-suspend in 100 uL of staining buffer + 0.1% permeabilizer.
6. Add appropriate amount of each antibody (eg. 1 test or 1 ug per sample, as experimentally determined).
7. Mix well and incubate at room temperature for 30 minutes- 1 hour. Gently mix samples every 10-15 minutes.
8. Following the primary/conjugate incubation, add 1-2 mL/sample of staining buffer +0.1% permeabilizer and centrifuge for 1 minute at 400 RCF.
9. Wash twice by re-suspending cells in staining buffer (2 mL for tubes or 200 uL for wells) and centrifuging at 400 RCF for 5 minutes. Discard supernatant.
10. Add appropriate amount of secondary antibody (as experimentally determined) to each sample.
11. Incubate at room temperature in dark for 20 minutes.
12. Add 1-2 mL of staining buffer and centrifuge at 400 RCF for 1 minute and discard supernatant.
13. Wash twice by re-suspending cells in staining buffer (2 mL for tubes or 200 uL for wells) and centrifuging at 400 RCF for 5 minutes. Discard supernatant.
14. Resuspend in an appropriate volume of staining buffer (usually 500 uL per sample) and proceed with analysis on your flow cytometer.

Immunocytochemistry Protocol

Culture cells to appropriate density in 35 mm culture dishes or 6-well plates.

1. Remove culture medium and wash the cells briefly in PBS. Add 10% formalin to the dish and fix at room temperature for 10 minutes.
2. Remove the formalin and wash the cells in PBS.
3. Permeablize the cells with 0.1% Triton X100 or other suitable detergent for 10 min.
4. Remove the permeablization buffer and wash three times for 10 minutes each in PBS. Be sure to not let the specimen dry out.
5. To block nonspecific antibody binding, incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
6. Add primary antibody at appropriate dilution and incubate overnight at 4C.
7. Remove primary antibody and replace with PBS. Wash three times for 10 minutes each.
8. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
9. Remove secondary antibody and replace with PBS. Wash three times for 10 minutes each.
10. Counter stain DNA with DAPi if required.
Immunohistochemistry-Paraffin Embedded Sections

Antigen Unmasking:
Bring slides to a boil in 10 mM sodium citrate buffer (pH 6.0) then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench-top for 30 minutes (keep slides in the sodium citrate buffer all the time).

Staining:
1. Wash sections in deionized water three times for 5 minutes each.
2. Wash sections in PBS for 5 minutes.
3. Block each section with 100-400 ul blocking solution (1% BSA in PBS) for 1 hour at room temperature.
4. Remove blocking solution and add 100-400 ul diluted primary antibody. Incubate overnight at 4 C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul HRP polymer conjugated secondary antibody. Incubate 30 minutes at room temperature.
7. Wash sections three times in wash buffer for 5 minutes each.
8. Add 100-400 ul DAB substrate to each section and monitor staining closely.
9. As soon as the sections develop, immerse slides in deionized water.
10. Counterstain sections in hematoxylin.
11. Wash sections in deionized water two times for 5 minutes each.
12. Dehydrate sections.
13. Mount coverslips.

[[URL:https://www.novusbio.com/products/glut1-antibody_nb110-39113]][[Caption… Antibody]]
Western Blot Protocol

1. Perform SDS-PAGE on samples to be analyzed, loading 40 ug of total protein per lane.
2. Transfer proteins to membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain according to standard Ponceau S procedure (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot.
5. Block the membrane using standard blocking buffer for at least 1 hour.
6. Wash the membrane in wash buffer three times for 10 minutes each.
7. Dilute primary antibody in blocking buffer and incubate 1 hour at room temperature.
8. Wash the membrane in wash buffer three times for 10 minutes each.
9. Apply the diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturers instructions) and incubate 1 hour at room temperature.
10. Wash the blot in wash buffer three times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturers instructions.

Note: Tween-20 can be added to the blocking or antibody dilution buffer at a final concentration of 0.05-0.2%.

FAQs for Glut1 Antibody - BSA Free

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  • Q: Can you tell me what, if any, antigen retrieval method was used for the staining of paraffin tissue sections with GLUT1 Antibody (NB110-39113)?

    A: We recommend the use of HIER (heat-induced epitope retrieval) with 0.01 M sodium citrate buffer, pH 6.0 at 100C for 20 minutes in a steamer or pressure cooker.

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