Anti-LAMP2 antibody [GL2A7] - Lysosome Marker (ab13524)

What is the epitope of ab13524, LAMP2 antibody [GL2A7]?

ANSWER:

Thank you for your enquiry.

As far as we know, the epitope of ab13524 is not determinded yet.

I hope this information helps you, and please do not hesitate to contact us if you have further questions.

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER R580318

DESCRIPTION OF THE PROBLEM There was no staining in the cytoplasm (where lysosomes are suppose to reside), but non-specific staining of the nucleus.

SAMPLE Mouse fibroblasts

PRIMARY ANTIBODY One hour at 1:100 dilution.

DETECTION METHOD Fluorescence

POSITIVE AND NEGATIVE CONTROLS USED Using the same cells during the same experiment, I used a different primary antibody with the same secondary antibody. Staining was positive.

ANTIBODY STORAGE CONDITIONS The anti-Lamp2 antibody was received, placed in a refriderator, and used within one (1) week.

FIXATION OF SAMPLE Paraformaldehyde

ANTIGEN RETRIEVAL No

PERMEABILIZATION STEP Saponin

BLOCKING CONDITIONS One hour using 10% goat serum

SECONDARY ANTIBODY One hour incubation with Cy2-conjugated anti-rat antibody.

HAVE YOU RUN A "NO PRIMARY" CONTROL? No

ADDITIONAL NOTES First, I am very experienced in performing immunofluorescence microscopy. For this particular experiment (using both anti-Lamp2, ab13524 and anti-ABCA1, ab18180), I used several dilutions of these primary antibodies (1:50, 1:100, 1:200 dilution) along with a primary antibody known to work. I then applied secondary antibody (Cy2-conjugated to an antibody that specifically recognizes the primary antibody). For both these antibodies, there was no visible staining in the cytoplasm, with faint staining of the nucleus. Previous "No Primary" control using the secondary antibodies did not label the nucleus. In brief, I suspect that these two antibodies are non-specific. I would really appreciate sending these vials of antibody back to Abcam, and getting a refund.

ANSWER:

I'm sorry to hear you are having a problem with ab13524 and ab18180.

I think the problem you are experiencing with the LAMP2 antibody may be due to the fixative, the antibody works only with methanol fixed cells or PLP (paraformaldehyde-lysine-periodate) but not with paraformaldehyde alone (my apologies for the error in the image which had omitted this detail, this has been corrected immediately). I would also suggest trying a longer incubation time with more dilute antibody (e.g 1:200-1:500) as this will maximise slow but targeted binding of the antibody to the LAMP2 protein.

It is difficult to know what the problem with the ABCA1 antibody staining could be due to as we have not tested this antibody in immunocytochemistry, only on paraffin sections. I would recommend to try different fixatives as they can make a big difference between various antibodies and to try a stronger permeabilising agent (I use 0.3% tritonx100 in my antibody dilution buffers as well as the blocking dilution buffer). Similarly to ab13524 you could try incubating longer in more dilute antibody and in my experience I prefer "stronger" secondary antibodies such as Cy3 or better, Alexa antibodies as they are brighter and hence give nicer signal.

If you still experience problems with the LAMP2 antibody with those recommendations please do not hesitate to contact me again and I can certainly offer you a refund for this product if you purchased the antibody in the last 90days, as it is guaranteed to work in ICC,

Unfortunately the newly shipped antibody shows the same results as its first delivery, meaning that we do not have any staining whatsoever. We have performed additional optimization steps includiong ice-cold acetone fixation without any effect. In the previous e-mail you state that the Ab was tested for ICC not for IHC, we do not perform IHC, but ICC under the same staining conditions as in the papers cited by Abcam on its www. Would you consider refund as a suitable solution for this matter?

ANSWER:

Thank you for your reply.

I am very sorry to hear that even the replacement vial failed to work. Looking at our order record in the computer system, I can see that we have sold a lot of vials of this particular product and our customer feedback indicates that we do not have serious complaint with it.

Since you are not satisfied with this antibody, for your request I will arrange a refund with my colleagues at the Account Department.

We apologize for any inconvenience caused. If you need anything further or any help in future then please let us know.

Do the antibodies bind to intracellular or extracellular domains?

ANSWER:

Thank you for your recent phone call regarding the location of the epitopes recognized by ab2733, ab2900 and ab13524.

As discussed on the phone ab2733 is known to bind the extracellular domain of the Mannose 6 Phospate Receptor. For ab2900 the immunogen sequence was found to be located at the C terminus of EEA1. We have been unfortunately unable to find out exactly where the epitope recognised by ab13524 is located as this has not been mapped. However, an image illustrating the staining pattern has been added to our datasheet and I hope this will help you. Since this antibody will label the presumptive lysosomes and late endosomes in cells that have been permeabilized with saponin this suggests that permeabilization is necessary and hence the epitope is likely to be on the intracellular side.

Please let me know if you need further information,

BATCH NUMBER 94267 ORDER NUMBER 72078

DESCRIPTION OF THE PROBLEM No staining

SAMPLE mouse fibroblasts

PRIMARY ANTIBODY ab 13524 diluted in 5% bovine serum albumine in PBS and finally 1:10, 1:25, 1:100, 1:200, different incubation times (4?C overnight, 37?C 1 hour)

SECONDARY ANTIBODY donkey anti rat IgG Alexa 488 labelled usual (1:1000) optimized for a number of other applications 1 hour 37?C

DETECTION METHOD fluorescent Nikon Ecclipse E800 equipped with C1 confocal head

no fluorescence visible in the pattern expected.

POSITIVE AND NEGATIVE CONTROLS USED Negative controls were performed by separate omission of both antibodies, positive controls were not performed due to totally negative results on the sample presumably positive and the necessity for optimization procedures. positive controls for secondary Ab were performed and were working OK

ANTIBODY STORAGE CONDITIONS according to the manufacturers recommendations, 4?C short term for aliquoting and -80?C for further storage.

FIXATION OF SAMPLE two alternative protocols cold Methanol 10 min / 4% paraformaldehyde 4?C 5 min

ANTIGEN RETRIEVAL none performed, we have never used these approaches due to very good quality material (cell line). Our extensive staining experience with cell lines including use of a number of them from Abcam never included this step.

PERMEABILIZATION STEP use of saponin 0,05% and/ or 0,5% Triton X-100, and their combination, with previous Methanol fixation

BLOCKING CONDITIONS 5% BOFES in PBS - 30 minut

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 15-20 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? No

WHAT STEPS HAVE YOU ALTERED? every (dilution of Abs, incubation temperaturature and duration, fixation, permeabilzation) in several reasonable variants everything with regard to your datasheet and published works.

ADDITIONAL NOTES Purchase order number was 2054000158 (could not fit into your pull down menu). We have consulted published articles (listed in the datasheet) that used the Ab previously and changed the protocol according to them even with dilutions 1:10 of primary, that were used previously despite your datasheet suggests 1:100. In one of the permeabilzation steps we have acquired quite clear nuclear staining, which is not specific according to declared Ab properties (photographs might be sent upon request). We have searched the possibilities of the use of another Ab clone, unfortunately this the only one suitable for our work. We have quite extensive experince in immunofluorescence and immunohistochemistry of lysosomal compartment epitopes (refer to our www and the list of publications - http://udmp.lf1.cuni.cz/web2/uvod/udmp.htm) We have ordered repeatedly from Abcam (GeneAhe - orders) this the first problem we have encountered with your products. We would very much prefer the replacement of the antibody with respect to previously published results. We are not aware of any obvious mistake on our side. Optimisation steps were very laborious and time comsuming not regarding the fact that we have actualy nearly ran out of the Ab due to low dilutions used. Thank you for your help. We are looking forward to positive reply.

ANSWER:

Thank you for your enquiry and for providing more information about your protocol. I am very sorry to hear that you are having problem with this antibody.

As the datasheet indicates, this antibody has only been tested for ICC but not for IHC.

I have read through your detailed protocol and it looks fine. However, I would suggest blocking the non-specific binding sites with 1 or 3% BSA for 30 min and not with 5% BOFES. The other suggestion would be to fix the samples in ice-cold acetone for 5 or 10 min to see if the signal comes up with a different fixative or not.

I have placed a new order for you one vial for replacement (free of charge) from a different batch: 103823. For your information, the new order number is 79621.

I hope this will work. Please do let me know how you are getting on with the new vial.