Anti-Syndecan-1 antibody (ab60199)

Thank you for sending the additional details of your protocol and the image of your blot.

Based on your results, it appears that the band between the top two markers is showing the expected expression pattern in your WT, KO, and knockdown samples. After doing some research, it is definitely possible that the protein could be running around 120 kDa or higher. According to the UniProt database, Syndecan-1 has a number of heparan sulfate and chondroitin sulfate modifications. These particular glycosylations can each range in molecular weight from 10-100kDa. Varying levels of glycosylation could account for some of the multiple bands you are observing. Additionally, if the protein is too heavily glycosylated, it may not run or transfer properly, causing you to lose some specific signal.

Unless you are specifically studying these glycosylations, removing them may help give you a clearer image. I would recommend pre-treating your samples with either Trifluoromethanesulphonic acid (which removes all N and O-linked glycosylations), or a mix of enzymes such as PNGase Fand O-Glycosidase.

In addition to removing the glycosylations, increasing your blocking time may help to improve your results. Blocking overnight will help to reduce the background staining if it is caused by non-specific binding rather than glycosylation. Given that the signal you are observing is strong, I would reduce the primary antibody incubation time to 1-3 hours at the same dilution of 1:2500.

I hope this helps, please let me know if you need any additional information or assistance.

Thank you for your response.

Incubation with the primary antibody either at 4 degC overnight or at RT for an hour in Western blot application should have the same results. However, depending on how much time you have for the incubation and whether you have other jobs/commitments to do in the lab, sometimes it is more convenient to perform overnight incubation at lower temperature (4 degC) but it is up to you. I agree with you that for consistency, it is worth performing the experiments under the same conditions.

I hope this helps and if I can assist further, please do not hesitate to contact me.

Thank you for your email.

I believe my colleague T. just send you the reply to your first question.

Regarding your second question, unfortunately, I have to confirm that we do not know if this antibody is suitable for IF yet. This has not been tested by us.

May I suggest to try it since you own the antibody already.

Protocols can be found here:

https://www.abcam.com/index.html?pageconfig=popular_protocols

https://www.abcam.com/index.html?pageconfig=resource&rid=11462

https://www.abcam.com/index.html?pageconfig=resource&rid=11383

https://www.abcam.com/index.html?pageconfig=resource&rid=11417

We would like to encourage you to let us know about your results by submitting an Abreview. Abreviews provide valuable information for other scientists and us on how the antibody works with different samples and in different applications. you will earn Abpoints with each Abreview that can be redeemed as discount on future purchases or Amazon gift vouchers.

I hope this information is helpful and wish you success with your research.

Thank you for your enquiry and your interest in our products.

This antibody has been tested for Western blot application by ECL detection system. Incubation for 1 hr @ RT or overnight @ 4 deg C should be fine. You may find some useful general information about WB on this site:

https://www.abcam.com/index.html?pageconfig=resource&rid=11375

https://www.abcam.com/index.html?pageconfig=resource&rid=11404#C5

As the datasheet indicates, with cell lysate of ScN2a cells, the antibody gave a clear and strong signal at a dilution of 1/2000. However, the final working dilution depends on many factors such as expressed levels of the target, sample preparation, loading amount, detection system so optimization is necessary. You may need to check different dilutions (1/5000, 1/2000, 1/1000 etc) to see which is the most optimal in your assay. Since you will be applying a more sensitive detection (SuperSignal West Femto) , you may need to dilute the antibody further. My advice would be to test and make sure that the secondary antibody works properly with other primary antibody.

If you need any further assistance in the future, please do not hesitate to contact me.

Thank you for contacting us.

Here are my suggestions for the targets you mentioned:

SMC alpha actin - ab5694
Calponin - ab46794
Myosin heavy chain - ab683 (heavy chain II) or ab682 (heavy chain I)
SM22 - ab14106

The antibody introduction is at the link below. We do not have a PDF version.

https://www.abcam.com/index.html?pageconfig=resource&rid=11287


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

Thank you for your enquiry. The WB blot on our datasheet was prepared as follows: 3T3 cells grown to confluency were detached using trypsin/EDTA. These cells were spun down and washed with PBS, then resuspended in PBS at about 5-10 million cells/ml. An aliquot of the cells was directly added to the SDS-PAGE sample buffer, cooked and applied at about 10-20 ug/ml total protein. No heparitinase was used and you can see on the datasheet that Syndecan-I protein shows a good band on our western blot. However, the observed band size is larger than predicted due to haparan sulfate and chondroitin sulfate modifications. If there is anything else that I can help you with, please do not hesitate to contact me.

Thank you for your enquiry. I can confirm that this product is specific for Syndecan 1. Also, based on the immunogen sequence, the antibody will not likely react with Syndecan 2, 3 & 4, as the sequence homology is not significant. I hope this information will be helpful. If there is anything else that I can help you with, please do not hesitate to contact me.