Positive and Negative Controls

To reduce variables in any type of experiment, it is recommended to include both positive and negative controls in the experimental design. Negative controls are particular samples included in the experiment that are treated the same as all the others but are not expected to change from any variable in the experiment. The positive control sample will show an expected result, helping the scientist understand that the experiment was performed properly. Some controls are specific to the type of experiment being performed, such as molecular weight standards used in protein or DNA gel electrophoresis, i.e. SDS-PAGE or agarose gel electrophoresis. The proper selection and use of controls ensures that experimental results are valid and can save valuable time.

Loading Control Antibodies

Loading control antibodies mostly recognize housekeeping proteins in cells used in a scientific experiment and allow the verification of equal protein loading between samples. Ideal loading controls are expressed constitutively and at high levels with low variability between cell lines and experimental conditions.

Loading controls are essential for the interpretation of assays. In Western blot assays, the loading control should be at a different molecular weight than the protein of interest, as this allows the protein to be visually distinguishable. Loading control antibodies not only allow the verification of equal protein loading between samples in Western blot assays, but they also allow for identification of certain cell compartmentalization or cellular localization in immunofluorescence microscopy (IF) and immunohistochemistry (IHC). Rockland’s loading control antibodies are suitable in assays including ELISA, FLISA, Western blot, IF, and IHC.

Western blot of pERK1/2, ERK1/2. α-tubulin is used as a control to illustrate uniform protein loading.

Control Cell Lysates and Nuclear Extracts

Rockland offers control cell lysates and nuclear extracts for use on SDS-PAGE as standalone samples or in combination with antibodies in Western blotting experiments. Our ready-to-use whole-cell lysates and nuclear extracts are derived from cell lines or tissues using highly advanced extraction protocols to ensure high quality, protein integrity, and lot-to-lot reproducibility.

Lysates are generated from either whole cells, which contain cell membrane, cytoplasmic, and nuclear proteins, or nuclear extracts, which are predominantly proteins that originate in the nucleus. Control lysates may be from cells that are stimulated with insulin, doxorubicin, etoposide, nocodozole, TNFa, or EGF. Lysates are also available from normal animal tissue derived from primary organs such as liver, heart, and brain. Additionally, Rockland offers a variety of lysates that contain over-expressed proteins (tagged and untagged) that can serve as positive controls for antibody reactivity. All extracts are tested by SDS-PAGE using 4–20% gradient gels and immunoblot analysis using antibodies to key cell signaling components to confirm the presence of both high molecular weight and low molecular weight proteins.

Purified Proteins

Purified proteins or peptides are ideal as controls in flow cytometry, Western blot, and ELISA. Proteins can be used as loading controls in Western blot experiments or as titration agents in ELISA experiments. Rockland produces purified immunoglobulin proteins from a variety of species, often available by immunoglobulin class or as fragments of immunoglobulins. Peptides can be used to do competition assays or to be used in peptide arrays.

Low Endotoxin Controls

Low endotoxin control proteins are IgG preparations of control serum purified by protein A chromatography using a low endotoxin methodology. These controls are ideal in biological assays like neutralization experiments, ELISA, flow cytometry, and other assays. For neutralization assays, where antibodies to cytokines, interleukins, infectious disease, and growth factors may be used to block bioactivity, our low endotoxin IgG serve as ideal control proteins. Rockland offers purified, low-endotoxin mouse and rabbit IgG.

Low Endotoxin Controls: