Anti-L1CAM antibody [2C2] - Neuronal Marker (ab24345)

LOT NUMBER -- NOT SPECIFIED -- oRDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No staining SAMPLE mouse adrenal gland PRIMARY ANTIBODY ab24345 DETECTION METHOD IF POSITIVE AND NEGATIVE CONTROLS USED None used -- can you recommend a positive control? ANTIBODY STORAGE CONDITIONS 4 degrees FIXATION OF SAMPLE 4% PFA, 4 degrees, O/N ANTIGEN RETRIEVAL tried with no retrieval and with SDS PERMEABILIZATION STEP 3% BSA, 3% goat serum, 0.3% Triton X-100, 0.3M glycine in PBS BLOCKING CONDITIONS Same as permeabilization SECONDARY ANTIBODY Alexafluor goat anti-mouse IgG1 HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes dO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes ADDITIONAL NOTES Can you provide a recommended staining protocol for tissue immunofluorescence for this antibody? (eg fixative, blocking, etc)

ANSWER:

Thank you for contacting us. I am sorry to hear your experiencing problems with our antibody. Could you please let me know what type of sections you are using? As of now, fixed-frozen or paraffin-embedded sections have not been tested with this antibody. Only IHC with perfused-frozen sections (IC-FoFr) has been performed, with an antibody dilution of 1/300 to 1/1000. The optimal dilutions would need to be determined by each researcher. As positive control rat cerebellum could be used as the antibody was tested on such sections. This is the protocol that has been used with the rat sections for perfused frozen sections: The immunohistochemical analysis was performed on sagittal Vibratome sections of 7-d rat cerebellum. Rats were perfusion fixed with picric acid-paraformaldehyde-glutaraldehyde, and sections were stained with peroxidase-conjugated second antibody as described below. Tissue fixation and staining. Sprague-Dawley rats were anesthetized with sodium pentobarbital (6.5 mg/100 g body weight) and perfused through the left ventricle, first briefly with 0.1 M phosphate buffer (pH 7.4), followed by the picric acid-paraformaldehyde-glutaraldehyde fixative described by Somogyi and Takagi (Somogyi, P., and Takagi, H. (1982) Neuroscience 7, 1779-1783). The brain was removed and allowed to stand in fixative for 2h at room temparature before being placed at 4 C overnight. After washing several times with PBS, the cerebellum was sectioned with a Vibratome to 15-30 um thickness for light microscopy. All sections were washed with 50 mM TBS (pH 7.6) before antibody staining. Sections were washed for 30 min in TBS containing 0.5% H202, 3 x 15 min in TBS, followed by 1h in TBS containing 5% BSA, 1% normal rabbit serum, and 0.2% Triton X-100. They were then incubated overnight at 4C in TBS containing monoclonal antibody, 0.5% BSA, 0.1% normal rabbit serum, and 0.2% Triton X-100. After washing for 1 h at room temperature in several changes of TBS without Triton X-100, the sections were incubated for 1.5 h at room temperature in TBS containing 0.5% BSA, 0.1% normal rabbit serum, 0.2% Triton X-100, and peroxidase-conjugated rabbit anti-mouse immunoglobulins (1:200). The sections were then washed for 1 h in several changes of TBS without Triton X-100, followed by 12 min incubation with 0.05% 3,3'-diaminobenzidine/0.01% H202 and 3 x 10 min washes in TBS. Sections were mounted on gelatin-coated slides, dehydrated through a series of graded ethanols, cleared with xylene, and sealed in Permount. Please let me know if this protocol is helpful to you and gives the desired results. If not, please let me know as well. I wish you good luck and look forward to hear back from you.

I am seeing only the cleavage products on my gel, and no band for the full-length protein. Has this been seen before in testing?

ANSWER:

Thank you for your enquiry.

The originator of the antibody stated that you could see the cleavage products and no full-length protein in certain sample types, and recommended running a positive control. The positive controls recommended are rat cerebellum or nervous system tissue.

I hope this information helps. Please do not hesitate to contact us if you need anything further.

When should we expect an answer? Thanks,

ANSWER:

Thank you for your enquiry.

Further to correspondence with the source of this antibody I can tell you that the concentration of this antibody has not been measured. However, its concentration is estimated to be ~ 1 mg/ml.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

I ordered mouse monoclonal antibody against L1CAM (code: ab24345) and received the product on last Tuesday. I tried it out on IHC, but the immunoreactive cells didn't seem to be neuronal cells and the staining of antigen was located in the nucleus. Therefore, I performed a western blot on whole cell lysate to confirm the size of the antigen, and I got one dominant band with a size of 50 kDa aproximally. I was wondering why these would happen. Could you please check the product of the same lot that recognize the L1CAM antigen? Thanks a lot.

ANSWER:

Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody.

ab24345 detects a band of approximately 200 kDa. Cleavage products are observed at 60-80 kDa. I ask, could it be that your band at approximately 50KDa is the cleavage products stated?

ab24345 has been tested using IHC against free floating sections. We recommend a dilution range of 1/300 - 1/1000. It does sound particularly strange that you have been obtaining nuclear staining with this antibody.

I would greatly appreciate it if you could complete our technical questionnaire by clicking on the link below. This will better enable our technical team to determine the steps that you have taken to optimise this antibody for your IHC application. It would also be particularly useful if you could provide me with details of the LOT number and a representative image.

http://www.abcam.com/index.html?section=ihc&pageconfig=technical&intAbID=24345&mode=questionaire

I look forward to hearing from you.