Antibody Specificity in Your Hands

 

Negative Control Antigen Manipulation

The negative control antigen is the peptide used to immunize the animal (immunizing peptide). All polyclonal antibodies are shipped with the negative control antigen at no extra cost. It’s the “clear vial” you receive with the antibody.

Prior to releasing new lots of an antibody, our dedicated in-house staff performs appropriate experiments to ascertain that the new lot is as specific as the previous lot using the negative control antigen. Since it is highly probable that you have a different experimental setup, we provide you with the experimental tools to also determine the specificity of the antibody in your lab.

Using the negative control antigen is quite simple. After calibrating the conditions under which the antibody works, you can run a side by side experiment.

Here’s how:

For a 1:200 dilution, add to a 1.5 ml tube 20 µg antibody and 500 µl 1% BSA in PBS. To a second 1.5 ml tube, add 20 µg antibody, 20 µg negative control antigen (supplied with the antibody) and 500 µl 1% BSA in PBS. Incubate both tubes for 1 hr at room temperature. Transfer the content of each eppendorf to larger tubes and add 4.5 ml 1% BSA in PBS with 0.1% tween-20 and 0.05% NaN3 to each.  Add the content of each tube to its respective membrane for parallel experiments. Incubate overnight at 4֯C with gentle rocking.

The following day, discard the primary antibody solution and wash membranes 3 times with washing buffer for 15 minutes at room temperature. Incubate with secondary antibody diluted accordingly for 1 hour at room temperature with gentle rocking.

Proceed to detection.

Compare the results of antibody alone versus antibody with negative control antigen. The disappearance of the requested band confirms the specificity of the antibody.

 

How do YOU use the negative control antigen?

Examples of published work using the negative control antigen:


Figure 1. Expression of P2X7 receptor in mouse and human hippocampus.Western blot analysis of mouse (upper panels) and human (lower panels) hippocampal lysates using Anti-P2X7 Receptor Antibody (#APR-004). In both species tested, P2X7 receptor is clearly detected. In samples probed with the antibody preincubated with the negative control antigen (right upper and lower panels), no signal is observed for P2X7 receptor, showing that the antibody specifically detects P2X7. In an additional control experiment, Anti-P2X7 Receptor Antibody is preincubated with the Anti-P2X4 Receptor Antibody (#APR-002) negative control antigen. As observed, the negative control antigen for the P2X4 antibody does not block the Anti-P2X7 Receptor Antibody.Adapted from Jimenez-Pacheco, A. et al. (2016) J. Neurosci. 36, 5920 with permission of the Society for Neuroscience.



Figure 2. Expression of TRPV4 in mouse kidney cell line.Immunocytochemical staining of mouse mCCDcl1 kidney cells using Anti-TRPV4 Antibody (#ACC-034), (green). TRPV4 staining is completely abolished when the antibody is preincubated with the negative control antigen (right panels).Adapted from Li, Y. et al. (2016) PLoS ONE 11, e0155006 with permission of PLoS.



Figure 3. Expression of KISS1 receptor in various human cell lines.Western blot analysis of human plasmacytoma (INA-6) cells, human mesenchymal stem cell (MSC), and human breast cancer cell lysates using Anti-KISS1 Receptor (extracellular) Antibody (#AKR-001), (upper panels). The band observed is completely eradicated when the antibody is preincubated with the negative control antigen.Adapted from Dotterweich, J. et al. (2016) PLoS ONE 11, e0155087 with permission of PLoS.



Figure 4. Expression of CaV2.3 channel in mouse accessory olfactory bulb.Immunohistochemical staining of mouse accessory olfactory bulb sections using Anti-CaV2.3 (CACNA1E) Antibody (#ACC-006). A(i). CaV2.3 staining (green) is detected in mitral cells. A(ii). High-magnification of (i). A(iii). Note the absence of immunofluorescent staining when the antibody is preincubated with the negative control antigen.Adapted from Gorin, M. et al. (2016) J. Neurosci. 36, 3127 with permission of the Society for Neuroscience.



Figure 5. Expression of NaV1.6 in mouse VNO.Immunohistochemical staining of mouse vomeronasal organ (VNO) sections using Anti-NaV1.6 Antibody (#ASC-009). A. NaV1.6 staining (red) is strongly detected in knobs (arrows) and somata (brackets). NaV1.6 expression intensity declines from basal to apical extremities. B. Lack of immunostaining is observed when the antibody is preincubated with the negative control antigen.
Adapted from Bolz, F. et al. (2017) Front Neuroanat. doi: 10.3389/fnana.2017.00028. with permission of Frontiers in Neuroanatomy.