CD28 Overexpression Lysate
Novus Biologicals, part of Bio-Techne | Catalog # NBP2-04308
Key Product Details
Species
Human
Applications
Western Blot
Product Summary for CD28 Overexpression Lysate
CD28 Transient Overexpression Lysate
Expression Host: HEK293T
Plasmid: RC211318
Accession#: NM_006139
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.
Expression Host: HEK293T
Plasmid: RC211318
Accession#: NM_006139
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.
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.
TMW
23 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.
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 CD28 Overexpression Lysate
![Western Blot: CD28 Overexpression Lysate [NBP2-04308]](https://resources.bio-techne.com/images/products/CD28-Overexpression-Lysate-Adult-Normal-Western-Blot-NBP2-04308-img0001.jpg "Western Blot: CD28 Overexpression Lysate [NBP2-04308]")
Western Blot: CD28 Overexpression Lysate [NBP2-04308]
Western Blot: CD28 Overexpression Lysate (Adult Normal) [NBP2-04308] Left-Empty vector transfected control cell lysate (HEK293 cell lysate); Right -Over-expression Lysate for CD28.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: CD28
CD28 is the prototypical and best-characterized costimulatory molecule on T cells (4). Its signals are critical for optimal naive T cell activation, cytokine production, proliferation, and survival (4). In order to sustain T cell activation, CD28 will consolidate immunological synapse formation, increase cell cycle progression through upregulated D-cyclin expression, and aid in T cell survival by in inducing the expression of the anti-apoptotic protein Bcl-XL (5). CD28 is constitutively expressed on naive and central memory CD4+ and CD8+ cells (5). CD28 deficiency has a large impact on T cell responses including activation, proliferation, immunoglobulin (Ig) class-switching, and germinal center (GC) formation (6). CD28 is a critical regulator of autoimmune diseases and tolerance to solid organ transplants in human patients (6). The CD28 pathway plays a central role in immune responses against pathogens, autoimmune diseases, and graft rejection (7). CD28 engagement via antibodies augments the proliferation of T cells in response to immobilized anti-CD3 antibodies (8). Additionally, antibody engagement of CD28 can supply costimulation to T cells encountering APCs deficient in costimulatory ligands, such as CD80 and CD86, and prevents the resultant anergic state that otherwise occurs in the absence of costimulatory signaling (8).
References
1. Esensten, J. H., Helou, Y. A., Chopra, G., Weiss, A., & Bluestone, J. A. (2016). CD28 Costimulation: From Mechanism to Therapy. Immunity, 44(5), 973-988. https://doi.org/10.1016/j.immuni.2016.04.020
2. Carreno, B. M., & Collins, M. (2002). The B7 family of ligands and its receptors: new pathways for costimulation and inhibition of immune responses. Annual review of immunology, 20, 29-53. https://doi.org/10.1146/annurev.immunol.20.091101.091806
3. Ward S. G. (1996). CD28: a signaling perspective. The Biochemical journal, 318 (Pt 2), 361-377. https://doi.org/10.1042/bj3180361
4. Zhang, R., Huynh, A., Whitcher, G., Chang, J., Maltzman, J. S., & Turka, L. A. (2013). An obligate cell-intrinsic function for CD28 in Tregs. The Journal of clinical investigation, 123(2), 580-593. https://doi.org/10.1172/JCI65013
5. Evans, E. J., Esnouf, R. M., Manso-Sancho, R., Gilbert, R. J., James, J. R., Yu, C., Fennelly, J. A., Vowles, C., Hanke, T., Walse, B., Hunig, T., Sorensen, P., Stuart, D. I., & Davis, S. J. (2005). Crystal structure of a soluble CD28-Fab complex. Nature immunology, 6(3), 271-279. https://doi.org/10.1038/ni1170
6. Bour-Jordan, H., & Blueston, J. A. (2002). CD28 function: a balance of costimulatory and regulatory signals. Journal of clinical immunology, 22(1), 1-7. https://doi.org/10.1023/a:1014256417651
7. Krummel, M. F., & Allison, J. P. (1995). CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. The Journal of experimental medicine, 182(2), 459-465. https://doi.org/10.1084/jem.182.2.459
8. Luhder, F., Huang, Y., Dennehy, K. M., Guntermann, C., Muller, I., Winkler, E., Kerkau, T., Ikemizu, S., Davis, S. J., Hanke, T., & Hunig, T. (2003). Topological requirements and signaling properties of T cell-activating, anti-CD28 antibody superagonists. The Journal of experimental medicine, 197(8), 955-966. https://doi.org/10.1084/jem.20021024
Alternate Names
CD28
Gene Symbol
CD28
Additional CD28 Products
Product Documents for CD28 Overexpression Lysate
Product Specific Notices for CD28 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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