Anti-PSD95 antibody (ab12093)

regarding primary antibodies against the below neuroscience targets that cross-react with zebrafish. She would like to use the antibodies in ICC and WB. If there is no antibody available would it be possible to do sequence alignments to look for regions of homology and check whether we have any antibodies whose epitope in this homology region?

The targets are:

glutamate receptors-AMPA GluRI and GluR2 subunit and WMPA(not sure about the hand writing of the later)

post-synaptic density-95

synaptophysin

NMDA-NR2A/NR2B/WRI subunits

Thanks a lot!

ANSWER:

Thank you for contacting us.

We do not have anti GluR1 and GLuR2 antibodies that can be used for Zebrafish. We unfortunately do not have information of GluR1 and GLuR2 protein sequence of Zebrafish so I am sorry we can’t check the homology. However if you have sequence information then please send it to us we will then give you the cross reactivity information of antibodies currently we have in our catalogue.

For PSD95 the antibodies ab12093 and ab90426 have quite homologues immunogen sequence so these antibodies may cross react with Zebrafish protein also.

We do not have anti Synaptophysin antibodies which have sequence homology with Zebrafish protein. So we cannot recommend any product.

Anti NMDAR1; the immunogen sequence of antibody ab32915, ab77264 and ab28669 is quite homologous so these antibodies could be used against Zebrafish species also.

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

Thanks for your email. Unfortunately I am unable to give you more details about our samples. We have repeated the experiments with new material and there we could see only one line. Therefore it seems everything ok with the antibody, the protocol etc. I hope this information helps.

ANSWER:

Thank you for your rapid feedback, this information indeed helps.

Could you please consider submitting a review when you have some time and in return we will offer you 50 Abcam Loyalty points which you can use to redeem a variety of gifts (another 100 points will be offered for an image of your blots).

I wish you all the best with your research,

Follow up to previous correspondance: We finally solved the problem. It seemed to be our samples.

ANSWER:

Thank you for your e-mail, I am glad to hear you have solved your problem, would you mind letting me know what it was so I can close this enquiry?

Thank you in advance,

Thanks for responding so fast.

I have checked the lysis buffer and we have used the following one, recommended for 1D-SDS-PAGE for mouse brain tissue:

40mM tris, 5M urea, 2M thiourea, 4% chaps, 10mM DTE, 1mM EDTA, Protease inhibitor cocktail from Roche.

We did not use the RIPA buffer, since this is more appropriate for cells. I actually do not think that the composition of the lysis buffer will cause the appearance of three bands.

We worked constantly at 4°C, and we used a protocol from the web for the semi-dry western blotting, which is used for this antibody from a different company. I contacted this company and they ensured me that they detect only one band and that this protocol can not cause the appearance of three bands, which I also strongly believe. The protocol is as follows:

*Blot buffer: 192 mM glycine; 25 mM Tris-base; 20 % (v/v) methanol; 0.04 % SDS. *Blocking solution: 20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 5% (w/v) skimmed milk powder; 0.02 % sodium azide; 0.1 % Tween 20. *Washing solution: 20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.1 % Tween 20. *Substrat buffer for alkaline phosphatase: 100 mM Tris-HCl, pH 9.5; 100 mM NaCl; 5 mM MgCl2. *BCIP staining solution: 20 mg/ml in 100 % di-methyl formamide. *NBT staining solution: 50 mg/ml in 70% di-methyl formamide. *Staining solution complete: Substrate buffer containing 80 µl BCIP solution and 60 µl NBT solution per 10ml and is mixed freshly for each staining.

The protein sample to be examined is separated by SDS PAGE and transferred to a nitrocellulose membrane by semi-dry electro-blotting using ~0.8 mA/cm2 (~0.3 mA/inch2).

1.Rinse the membrane in water and incubate in blocking solution for 30 min on a lab shaker (gently rocking). 2.Replace with fresh blocking solution containing the primary antibody in the appropriate dilution and incubate for at least 2 h on a lab shaker. 3.Wash 3 - 4 times with blocking solution for 10 min each time. 4.Replace with fresh blocking solution containing the recommended secondary antibody (anti-mouse IgG, anti-rabbit IgG, resp.) in the appropriate dilution and incubate for at least 1 h on a lab shaker. 5.Wash 3 times with washing solution for 10 min each time. 6.Replace washing solution with substrate buffer and let equilibrate for 5 min. 7.Replace with fresh staining solution complete and develop for 15 - 30 min. Time can be shortened or extended, if signals are extremely strong or weak, resp. 8.Stop staining reaction by washing 3 times with H2O.

As you can see the protocol is the same as the one that you are referring to at your webpage.

To see the result of such a blot, I have attached an example. The molecular weight is not exactly.

The only logical explanation for me is that your antibody, since it is a polyclonal antibody, is not purified enough and therefore not very specific. Would it be possible for you to send us for no further charge a sample of a different batch, so that we can check this?

ANSWER:

My sincere apologies for the mistake in your e-mail address.

I agree with you that the cocktail of inhibitors from Roche you use is the equivalent to my recipe of protease inhibitors, I am however unfamiliar with the chemicals 5M urea, 2M thiourea, 4% chaps and 10mM DTE in a lysis buffer, they seem to be reducing agents (definitely urea) and reducing agents should be present only in the loading buffer rather than the lysis buffer, adding the loading buffer at the last minute prior to boiling. If you use the same buffer for lysing and loading the various bands could be degradation products. My apologies if I am misunderstanding this, could you therefore clarify if this is the case?

The other change I would recommend is the presence of milk in the antibody dilution buffer which can give non specific bands in some rare cases unfortunately (especially with proteins which are phosphorylated), so I would try 5% BSA blocking and incubation of the primary antibody in TBST only.

I am looking at the original files from our collaborator who tested the antibody for us and what I find very interesting is that the band he shows is much larger and darker than your two bands, I am therefore wondering if the antibody at high concentration gives a "fat band" rather than two thin ones. I have worked on ERK in the past and if ERK1 and ERK2 were run and the bands were very close together they would appear as one large "merged" band, very much like the image we have. I have written to our collaborator to find out if he even tested with more separation and could see two bands and if it is possible that PSD95 comes as two forms? I doubt that a phosphorylation could cause a large shift in MW detectable by WB, but he may be able to shed some light into this.

I'm sorry for the delay in finding out this information and would appreciate if you could let me know about your buffer so I can rule out this potential problem,

Thank you in advance, I look forward to hearing from you,

The customer detects 3 bands with ab12093 rather than 1, in mouse brain extract.

ANSWER:

I'm sorry that you have been waiting for technical support with ab12093.

Here is the recommended lysis buffer to extract PSD95 from mouse brain:

RIPA buffer: 50mM Tris HCl pH=8 150 mM NaCl 1% NP-40 0.5% sodium Deoxycholate 0.1% SDS

inhibitors: Aprotinin 2µg/ml, Leupeptin 5-10 µg/ml, Anti-pain 2-10 µg/ml, Pepstatin A 1µg/ml, Na-fluoride 5-10 mM, Na-vanadate 1mM, PMSF 1mM. Please make sure the samples are kept at 4C at all times before the boiling in reducing loading buffer. Dissection should also be on ice and very rapid to decrease degradation.

I would recommend also trying a different blocking buffer: try 5% BSA in TBST for 1hr at RT, then rinsing the membrane gently in TBST and incubating the primary antibody at 4C overnight in TBST (incubate the secondary in TBST too).

Please do not hesitate to contact me again if you need further assistance,