GAPDH Overexpression Lysate

Novus Biologicals | Catalog # NBL1-10967

Novus Biologicals
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Key Product Details

Species

Human

Applications

Western Blot

Product Summary for GAPDH Overexpression Lysate

GAPDH Transient Overexpression Lysate


Expression Host: HEK293T

Plasmid: RC202309

Accession#: NM_002046

Protein Tag: C-MYC/DDK

You will receive 1 vial of lysate (100ug), 1 vial of empty vector negative control (100ug), and 1 vial of 2xSDS sample buffer (250ul). Each vial of cell lysate contains 100ug of total protein (at 1 mg/ml). The 2xSDS Sample Buffer consists of 4% SDS, 125mM Tris-HCl pH6.8, 10% Glycerol, 0.002% Bromophenol blue, 100mM DTT.

Product Specifications

Application Notes

This product is intended for use as a positive control in Western Blot. Overexpression of the target protein was confirmed using an antibody to DDK (FLAG) epitope tag (NBP1-71705) present on the protein construct.

Each vial of cell lysate contains 100ug of total protein which should be sufficient for 20-50 reactions. Depending on over-expression level, antibody affinity and detection system, some lysates can go as low as 0.1 ug per load. We recommend starting with 5ug of cell lysate. Add an equal amount of cell lysate and 2X SDS Sample buffer and boil the SDS samples for 10 minutes before loading.

Theoretical MW

36 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.

Type

Overexpression

Scientific Data Images for GAPDH Overexpression Lysate

Western Blot: GAPDH Overexpression Lysate [NBL1-10967]

Western Blot: GAPDH Overexpression Lysate [NBL1-10967]

Western Blot: GAPDH Overexpression Lysate [NBL1-10967] - (Adult Normal) [NBL1-10967] Left-Empty vector transfected control cell lysate (HEK293 cell lysate); Right -Over-expression Lysate for GAPDH. Theoretical molecular weight: 36 kDa.

Formulation, Preparation, and Storage

Formulation

RIPA buffer

Concentration

The exact concentration of the protein of interest cannot be determined for overexpression lysates. Please contact technical support for more information.

Shipping

The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.

Storage

Store at -80C. Avoid freeze-thaw cycles.

Background: GAPDH

Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) is a ubiquitous enzyme involved in glycolysis, converting glyceraldehyde-3-phosphate into 1,3 diphosphoglycerate. This constitutively expressed, homotetramer protein can be found in the nucleus and cytoplasm, and the monomer has a theoretical molecular weight of 36 kDa. Known as a housekeeping gene, GAPDH routinely serves as a control for real-time PCR (RT-PCR) and as a loading control for Western Blots (1-3). In addition to its role in glycolysis, GAPDH has multiple cellular functions including membrane trafficking, transcription activation, apoptosis, DNA repair, and has been implicated in various pathologies such as cancer and neurodegenerative diseases including Alzheimer's and Huntington's (4,5).

References

1) Barber RD, Harmer DW, Coleman RA, Clark BJ. (2005) GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues. Physiol Genomics. 21(3):389-95. PMID: 15769908

2) Jia Y, Takimoto K. (2006) Mitogen-activated protein kinases control cardiac KChIP2 gene expression. Circ Res. 98(3):386-93. PMID: 16385079

3) Godsel LM, Hsieh SN, Amargo EV, Bass AE, Pascoe-McGillicuddy LT, Huen AC, Thorne ME, Gaudry CA, Park JK, Myung K, Goldman RD, Chew TL, Green KJ. (2005) Desmoplakin assembly dynamics in four dimensions: multiple phases differentially regulated by intermediate filaments and actin. J Cell Biol. 171(6):1045-59. PMID: 16365169

4) Sirover MA1. (1999) New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase. Biochim Biophys Acta. 1432(2): 159-84. PMID: 10407139

5) Tristan C, Shahani N, Sedlak TW, Sawa A. (2011) The diverse functions of GAPDH: views from different subcellular compartments. Cell Signal. 23(2):317-23. PMID: 20727968

Long Name

Glyceraldehyde-3-phosphate Dehydrogenase

Alternate Names

G3PDH

Gene Symbol

GAPDH

Additional GAPDH Products

Product Documents for GAPDH Overexpression Lysate

Certificate of Analysis

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

Product Specific Notices for GAPDH Overexpression Lysate

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

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FAQs for GAPDH Overexpression Lysate

Showing  1 - 5 of 8 FAQs Showing All

    • A: We offer sample sizes for many of our GAPDH loading control antibodies including NB300-221.

    • Q: I need a suitable loading control antibody that I can use in western blot of rat nerve extracts, which do you recommend?
      A: This GAPDH antibody (NB300-221) is a good choice for a loading control antibody and has been cited in over 180 publications and used on rat lysates with good results.

    • A: Our GAPDH antibody makes a good loading control because GAPDH is  a housekeeping gene essential to metabolism that is globally expressed in most tissue types and cell lines.

    • Q: I wanted to know which of the two housekeeping genes B-actin or GAPDH can be used for best results. I intend to do an experiment to determine expression of IL-6 and TNF-alpha in liver and kidney tissues.
      A: For homogenized tissue, beta-actin and GAPDH are both fine.

    • Q: I'm looking for a loading control between 24kDa - 65kDa for use in simple western, we tried an actin antibody  we already had but it didn't work. What can you recommend?
      A: Our GAPDH antibody NB300-221 has been validated in simple western and detects around 35 kDa so I think it would be a good choice for a simple western loading control antibody based on your size requirements.

    • Q: We need a loading control antibody for WB working with HeLa cell line, will this GAPDH antibody work for that?
      A: GAPDH is expressed in HeLa cell lines but whether or not it is the best option for a loading control antibody depends on the size of the protein you are interested in. GAPDH antibody is a good choice for low to mid MW targets, as GAPDH detects around 35 kDa.

    • Q: We received GAPDH antibody NB600-502 and there was only about 15µl of antibody in the tube. I was under the impression this should have been 100µl.
      A: The unit size of this product is 0.1 mg with a concentration of 6.7 mg/ml. Hence the volume should be no less than 14.9 ul. 

    • A: This GAPDH antibody is raised in mouse so you would want to use an anti-mouse secondary with the dye of your choice.
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