Anti-Glycine antibody (ab9442)

Thank you for contacting Abcam.

1. Bovine calf serum rather than goat serum should work to sufficiently block non-specific binding.

2) As stated on the datasheet, this antibody is tested and guaranteed to react with rat Glycine via IHC-FoFr, IHC-Fr, ICC, Immunomicroscopy. Thus, our AbPromise guarantee would not apply to the mouse retina slices that you are mentioning.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

Thank you for contacting us. We have anti glycine or anti- proline antibodies in our catalogue. The catalogue numbers are ab9344, ab9345, ab37067, ab9442 and ab37087. We unfortunately do not have antibodies available specifically raised against proline-glycine-proline short peptide. I would like to recommend checking the Biocompare website which has an excellent antibody search facility that includes many suppliers. The links are: www.biocompare.com http://www.biocompare.com/ProductCategories/2045/Antibodies.html Elastin proteins also have repeating units of proline and glycine residues. So probably monoclonal antibodies against part of Elastin will be helpful e.g. ab97963 and ab51599. Although these do not exactly match however it will help to widen your search. I hope this information is nevertheless helpful to you. Please do not hesitate to contact me if you have any further questions in this regard.

Thank you for your enquiry. This product is sold as whole antiserum, not as purified antibody. An exact concentration is not available. In general, we estimate that the total Ig concentration in serum is approx. 10 mg/ml, and the specific antibody component is approx. 10% of this.

Thank you for your reply and for your interesting comment. We have looked at your article very carefully. This is a 4% PF fixation which does not cross-link tissue. That is why the penetration is better - but not good. Most anti-glycine antibodies absolutely require glutaraldehyde, since the target is so small, the concentration low and the risk of loss is high. Our IgG is specifically designed for glutaraldehdye and resin sections - we never sell it as a general PF IgG for frozen sections. Anti PF-glycine IgGs are not commercially available yet. Regarding the GABA ICC: The GABA signals are typical of frozen/vibratome sections and they are not what we would call good. Clearly they were adequate to answer the question at hand but do not represent the current standard for small molecule detection fidelity, coverage or signal strength. They represent incomplete coverage, non-quantitative signals, as evidenced by the failure to detect nuclear GABA levels which are usually equal to somatic levels. The immunocytochemistry reveals the presence of some, but clearly not all spinal GABA+ cells (only those with cut surfaces emerging somewhere on the sections). The mean levels of glycine in most glycinergic neurons is about 1 mM (partly limited by the synthetic mechanisms for glycine) and that GABA levels in GABAergic neurons are 5-10 fold higher. This means that glycine signals will be almost a log unit weaker that the GABA images you show, and this will be invisible with FITC under the best of conditions. We do have customers who have good qualitative glycine signals in frozen sections - but again - with great difficulty due to the poor penetration of the IgGs. Resin sections always work. Please do let us know how you are getting on with this product. If you are still experiencing problem, do not hesitate to contact us again. We will try to do our best to solve the problem a soon as possible.

Thank you for your enquiry and for providing more details of your protocol. As we understand from your e-mail, the main problem is that you have no glycine signals. This is very likely a technical failure. Reading through your protocol, it is clear that you have applied a normal IHC method. Here we would like to make some suggestions and comments: 1. Do not use frozen or vibratome methods, use resin sections instead. In general, amino acid antibodies will not work in glutaraldehyde cross-linked frozen or vibratome sections. This has been well-known since the 1980s. 2. Before proceeding, please do take a look at the special “Protocol” section on the top panel of the on-line datasheet of this product. Here you will find a detailed protocol for this product 3. Do not use immunocytochemistry method on small molecules and never use frozen or vibratome sections for small molecules. 4. Don't use FITC – the signal can be very low. We would recommend using Alexa red or Cy3 instead. Please remember that glutaraldehyde (which is essential for trapping small molecules) causes massive autofluorescence, often brighter than FITC. No wonder why you couldn't detect any signal. We also recommend HPI with silver detection. 5. You may also need to consider the fact that even glycinergic neurons have rather low levels of glycine - and it quickly disappears in ischemia. Are you certain that your spinal cord samples are not ischemic? 6. Confocal visualization is not necessary. If you want 3-D thick section visualization of glycine, remember that no one has published such material because it is virtually impossible to do so. We hope that this information will be useful for you.

Dear George 1) This antibody should work with 0.1% glutaraldehyde fixation using the High Performance Immunocytochemistry protocol listed with the antibody on the Abcam website. 2) I have had a search on the web and I have learned from this that glutaraldehyde autofluorescence can often be significantly reduced by washing the fixed material with 0.1% sodium borohydride in PBS. 3) I am afraid that we can't supply small sample volumes. However, our policy at Abcam is that if an antibody does not work as specified on the datasheet, we offer a replacement or reimbursement. We also offer a USD20 Amazon gift voucher to researchers who feedback information about our products. I hope this helps. Please feel free to get back to us with any further questions. Regards Michael