The QC Max™ range of antibodies is a well characterized and cited group of products. QC Max™ antibodies have been Quality Controlled for Western blots, immunocytochemistry, ELISA, immunoprecipitation and species specificity. That makes them some of the most highly characterized antibodies available to the research scientist.
For more information on the specifics about the antibodies, please click on the appropriate group of antigens below.
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At Cytoskeleton, we understand that many antibodies are suspect and have uneven quality. The QC Max series of four tests were created to give scientists the best picture of an antibody’s performance in different situations. The four QC tests are 1) Western blot, 2) Immunofluorescence in situ, 3) ELISA and 4) Immunoprecipitation. These tests are important to determine specific, affinity and antigen conformation recognized by each product which are critical factors in determining the spectrum of uses the reagent can be used for. The importance of performing these tests to fully characterize an antibody are described below.
1) Western blot
Western blot gives great information about specificity (the ability to detect only the antigen), and some information about the affinity. If the antibody can detect one antigen band in a lane which is loaded with 50 µg of extract then it is considered specific. If it can detect 1ng (50 femto-moles) of antigen using chemiluminescence detection it is considered high affinity.
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Figure 1. Western blot analysis of anti-Cdc42 antibody. Recombinant small G-proteins and tissue extracts were separated by SDS-PAGE and transferred to PVDF membrane. ACD03 was diluted to 250 ng/ml (1:1000) in PBST plus 0.1% non-fat milk powder and Western analysis was as described in the Methods section. Lane 1; 25 ng Cdc42-6xHIS, Lane 2; 250 ng RhoA-6xHIS, Lane 3; 250 ng Rac1-6xHIS, Lane 4; 40 µg Swiss 3T3 cell extract, Lane 5; 40 µg bovine brain extract, Lane 6; 40 µg human platelet extract. Note: Cdc42-6xHIS runs at 25kDal whereas the native Cdc42 runs at 22kDal. |
2) Immunofluorescence in situ
Immunofluorescence in situ (IF) and its counterparts immunohistochemistry (IH), fluorescence activated cell sorting (FACS) and high content screening (HCS) are performed by staining live or fixed cells or tissues with a fluorescent or colorimetric marker. The importance of this technique to characterize antibodies is to confirm that the reagent can detect the antigen in its native environment. An antibody may detect the native conformation (e.g. with paraformaldehyde fixation) or denatured antigen (e.g. with methanol fixation) and hence these aspects must be presented in the datasheet. Another consideration is that the antigen may be masked by another molecule e.g. protein or DNA, and hence may not work in this application unless a disrupting agent (e,g, methanol, detergent or chaotropic agent) is added first. A pre-requisite of this technique is to be sure the Western blot is giving just one band in a lane containing cell extract.
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Figure 2. Immunofluorescence images of mouse Swiss 3T3 cells stained with Anti-Tubulin polyclonal antibody (Cat. # ATN02). Swiss 3T3 cells were grown to semi-confluency and fixed with methanol. Immunofluorescence staining using 2.5 µg/ml (1:200 dilution) ATN02 antibody is shown (red). The primary antibody was detected with a 1:500 dilution of anti-sheep rhodamine conjugated antibody. DNA (blue) was stained with 100 nM DAPI in PBS. Photograph was taken with a 100X objective lens. |
3) ELISA
ELISA is a quantitative technique to determine the amount of antigen in a sample. The technique is important in determining the affinity of an antibody to an antigen. Usually high affinity reagents have Kd’s = 10-9 M or higher.
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Figure 3: Each well of an ELISA plate was coated with 2 ng of RH01, then blocked with dry milk and probed with sequential dilutions of ARH03. Bound antibody was detected with HRP-secondary anti-mouse antibody and signals developed with OPD reagent. The OD490nm signals were plotted on the y-axis to create a binding curve. The Kd was calculated from the 50% full scale signal x-axis intercept |
4) Immunoprecipitation
Immunoprecipitation (Ippt) is a useful technique to determine binding partners in an extract. It is particularly in signal transduction research where regulated binding partners may differentially occur in the pellet sample when activated or de-activated. The antibody is usually bound to a bead and used as an affinity matrix to pullout the antigen and any associated proteins.
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Figure 4. Immunoprecipitation of human MKLP1 from HeLa extracts using AKIN06. Western blot of HeLa cell lysate (Lane 1), IP supernatant after immunoprecipitation (Lane 2), MKLP1 immunoprecipitated with AKIN06 (98 kDa, see arrow) eluted from Protein A beads (lane 3), and control immunoprecipitation reaction with normal rabbit IgG (lane 4). Lanes 3 and 4 also show IgG heavy chain (55 kDa, arrow head). The Western blot was probed with 500 ng/ml (1:500 dilution) of AKIN06 antibody.
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Cytoskeleton's antibodies products have been cited hundreds of times over the past decade. A select few are described here, for more citations on individual products please use the "Citations" tab on each individual product page.
Anti-pan actin: rabbit polyclonal (Cat. # AAN01)
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Klees, R. F., Salasznyk, R. M., Kingsley, K., Williams, W. A., Boskey, A. and Plopper, G. E. (2005). Laminin-5 induces osteogenic gene expression in human mesenchymal stem cells through an ERK-dependent pathway. Mol. Biol. Cell 16, 881-890.
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Anti-profilin: rabbit polyclonal (Cat. # APUF01)
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| Osiak, A. E., Zenner, G. and Linder, S. (2005). Subconfluent endothelial cells form podosomes downstream of cytokine and RhoGTPase signaling. Exp. Cell Res. 307, 342-353. |
Anti-kinesin heavy chain: rabbit polyclonal (Cat. # AKIN01)
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| Stegman, M. A., Goetz, J. A., Ascano, M., Jr., Ogden, S. K., Nybakken, K. E. and Robbins, D. J. (2004). The Kinesin-related protein Costal2 associates with membranes in a Hedgehog-sensitive, Smoothened-independent manner. J. Biol. Chem. 279, 7064-7071. |
Anti-RhoA: mouse Mab (Cat. # ARH03)
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| Khan et al. (2011). Geranylgeranyltransferase type I (GGTase-I) deficiency hyperactivates macrophages and induces erosive arthritis in mice. J Clin Invest doi:10.1172/JCI43758. |
Anti-alpha/beta tubulin: sheep polyclonal (Cat. # ATN02)
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Sanbe, A., Osinska, H., Saffitz, J. E., Glabe, C. G., Kayed, R., Maloyan, A. and Robbins, J. (2004). Desmin-related cardiomyopathy in transgenic mice: a cardiac amyloidosis. Proc. Natl. Acad. Sci. U. S. A. 101, 10132-10136.
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Question 1: What is the best RhoA antibody available?
Question 2: What is the best Rac1 antibody available?
Question 3: At what dilution should I use the anti-tubulin sheep polyclonal antibody?
Question 1: What is the best RhoA antibody available?
Answer 1: There are multiple options when choosing an antibody to detect small GTPases. Several factors must be considered: specificity, sensitivity, format compatibility, species cross-reactivity and consistency of signal. Cytoskeleton’s RhoA mouse monoclonal antibody (Cat. # ARH03) scores high marks on all of these parameters. It is specific to RhoA, meaning not only will it not detect Rac or Cdc42 proteins, but it is isotype specific as well. It will not detect RhoB or RhoC. It is also very sensitive and works well for both western blotting and immunochemistry. Of course there are other companies (e.g. Cell Signal Technologies) that also make good antibodies that are comparable to ours. There have been reports of lot to lot variability with some supplier’s RhoA antibodies. Cytoskeleton’s antibodies do not have batch to batch variability due to our stringent quality control process.
Question 2: What is the best Rac1 antibody available?
Answer 2: There are multiple options when choosing an antibody to detect small GTPases. Several factors must be considered: specificity, sensitivity, format compatibility, species cross-reactivity and consistency of signal. We have performed direct comparisons between our Rac1 specific mouse monoclonal antibody (Cat. # ARC03) and that of our competitors, and we have found that our Rac1 antibody is the only one that is truly specific for Rac1. Others detect either Cdc42, Rac2 or Rac3 proteins in addition to Rac1. Our Rac1 specific antibody works for both western blotting and immunochemistry.
Question 3: At what dilution should I use the anti-tubulin sheep polyclonal antibody?
Answer 3: All dilutions are noted in the datasheet. The actual dilution of antibody depends on the cell/tissue type and experimental format being used. For western blotting, we have tested and recommend using the antibody at a concentration of 500 ng/ml (1:1000 dilution). At this dilution, we detected tubulin in extracts of Drosophila S2 cells, Xenopus A6 cells, mouse Swiss 3T3 cells, rat NRK cells, human HeLa cells, and bovine brain. For immunofluorescence, we used the anti-tubulin antibody to detect tubulin in mouse Swiss 3T3 cells. The antibody was used at a concentration of 2.5 μg/ml (1:200 dilution) followed by incubation with a 1:500 dilution of anti-sheep rhodamine-conjugated secondary antibody. This information is found usually in the figure legends or within the “Product Uses” section of our antibody datasheets.
