Anti-Glycine antibody (ab9442)

Customer would like to know if we can offer anti proline-glycine-glycine or anti proline-glycine-proline antibodies.

ANSWER:

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.

Customer has lot# 116580 - is a concentration (at least an approximate) available?

ANSWER:

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 details of IHC protocol that could improve the labeling of Glycinergic cells.

I don''t want to switch to resin sections. We use vibratome sections. For your information, we can obtain a good labeling on small molecules using frozen sections and FITC (see Allain et al. Brain Resarch 1000: 134-137, 2004).

We will keep trying to make your antibody work even if it represents a high cost for our lab compared to our very small financial supports.

Thank you anyway but I would have preferred a financial compensation....

ANSWER:

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.

Message 1:

We ordered twice (on september 18th 2003 and march 03th 2004) 500 µl of the rabbit polyclonal glycine antibody ab9442. Total cost 664 € ! So far we have not been able to collect any selective labeling on spinal cord sections fixed according to the recommended protocol as well as according to other protocols (our modified protocols). We made so many unsuccessful trials using the recommended high concentration (1/100) that we had to reorder this product. And today we are still fighting to obtain a specific labeling. We have thus spent time, money and energy (one of my student who works on this project is becoming very disappointed) ! Conclusion : your company sells an antibody that is defective.

Message 2.: Here are the informations :

-The problem : very few staining

- Material tested : • Species : mouse • Cell line : no • Tissu: spinal cord slices

- fixative: 1% paraformaldehyde - 2.5 % glutaraldehyde made in PB 0.1 M

- Antigen retrieval step : NO • Enzymatic method • Heat mediated technique • Other

- blockade of the unspecific binding sites : YES with 2% BSA (bovin serum albumin)

- Primary antibody:

• specification : made in rabbit • dilution : 1:100 (recommended dilution) • incubation time: 48 hours at 4°C • wash steps: numerous (at least 4 times during 10 minutes)

- Secondary antibody: fluorescein anti-rabbit made in goat 1:200 during 2 hours at room temperature (this procedure works routinely in our lab with others primary antibodies)

- Which detection system did you use : FITC and confocal imaging

- Did you apply positive and negative controls along with the samples ? What do you mean ?

- Optimization attempts :

• How many times have you tried the IHC : more than 15 .... I don't want to know... • Do you obtain the same results every time : YES • What steps have you altered : we first tried on frozen sections (cut using a cryostat) and then switched to sections embedded in agar and cut with a vibratome. It seemed to help but I am not satisfied with the staining. I think that I should obtain more numerous stained cells in the spinal cord sections.

ANSWER:

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.

Three enquiries really: 1) could this antibody work with low glutaraldehyde, in order to do immuno at the fluorescent level? 2) if gluta is essential, how do you propose to reduce/eliminate autofluorescence due to glutaraldehyde? 3) do you small "sampling" volumes (ie 100microliters) and if so, what is the price (including P&P)?

Dr George Mentis

ANSWER:

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