Anti-TSG101 antibody [4A10] - BSA and Azide free (ab83)

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You can use any protease inhibitor, most common are Leupeptin and PMSF. These protease inhibitors should be dissolved in lysis buffer.

People tend to use protease inhibitors cocktails which have many enzymes in it. These can bought from Sigma, Roche or other vendors. Please use these in lysis buffer as per manufacturer's instructions.

Chemically denaturation generally does not affect primary structure, but it does cause degradation of the complex three-dimensional arrangements of the proteins structures e.g. secondary, tertiary, and quaternary. Most protein functions result from the three-dimensional arrangements of the proteins, so denaturation of such structures generally results in a loss of protein function. In western blot proteins gest denatured at high temperature with degradation of secondary structures. The primary structuresare still intact for antibodies to bind.

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

Thank very much for filling the questionnaire and for sending the image.

I have reviewed the protocol now and would like to point out few things that may be the cause of multiple bands;

- Protease inhibitors; You have mentioned that no protease inhibitors were used but these are necessary to use in lysis buffer. I would suggest using these and redoing the lysis.

- In our own lab we heat the samples in loading buffer at 100C for 10 minutes. I would suggest heating the samples for 5-10 minutes.

- We have observed a single band in Hela, Jurkat, HEK293 and A431 cell lines; these cell lines are easily available so lysates of these can be used as positive control.

- Try loading 20ug protein.

- Trying a no primary control will help to check the non specificity of secondary antibody.

There are two isoforms of this protein one is 44kDa and other is 32 kDa. Please click the link http://www.uniprot.org/uniprot/Q99816 for more info. In 32 kDa isoform amino acid 15-112 is missing. The antibody recognize the epitope between 167-374 so it is more likely that the antibody will detect both the isoform but the isoform should be expressed in the samples. We are however not sure if the isoform is expressed in monocytes.

The Abcam anti TSG1 antibody has been used in following publication http://www.nature.com/bjc/journal/v92/n2/full/6602316a.html. In Fig 1 duplet has been observed. This publication may provide further insight into you r research.

I hope these suggestions will help to improve the results. If however the results do not improve please contact me I will be happy to assist further.

Thanks you for your email.

There could be many reasons for this extra band. It could be an Isoform; TSG101 have 2 isoforms one is 31kDa nd other is 44 kDa. Two bands could be observed however it will depend on the lysates being used. http://www.uniprot.org/uniprot/Q99816

It could be non specific band, which can go away with simple protocol troubleshooting e.g.
- Using 70-80% confluent cells
- Using Fresh protease inhibitors
- Using fresh samples

We have used Hela, Jurkat, A431 and kidney cells while testing this antibody and we have observed no extra band which could means that the isoform is not expressed in cell line. I would recommend using the lysates of same as positive control.

I can provide further help however would need to know more about the protocol. I have attached a questionnaire with my email, please fill in and send it to me asap.

I hope these suggestions will help to improve the results.

Thank you for contacting Abcam.

Forthese antibodies I would recommend using reducing conditions and also initially using a 1/1000 primary antibody dilution, although you may to alter this concentration for subsequent western blots.

If there is anything else I can help you with, please let me know.

The datasheet was correct and the customer should have received 100ul, I apologise for this problem and have arranged a free of charge vial to be sent to you. Many thanks for your help with this matter,

Thank you for your email. I contacted the originator of this antibody who was able to provide me with the following information. "Concerning IHC staining, you may want to refer to the following publication for a detailed protocol: Zhu et at, Oncogene 22:3742 (2003). 1. The authors in the previously-referenced paper were using paraffin-embedded tissues (this particular group was looking at human tissues. I do not have an IHC reference for rat or mouse). 2. No antigen retrieval was necessary. 3. Following thorough washing in 0.015 m TBS (pH 7.6) and blocking with 20% normal rabbit serum, the antibody, at 1:100, was applied for 1 hr. 4. Sites of antIgen-antibody binding were detected using biotinylated rabbit anti-mouse Ig followed by peroxidase conjugated streptavidin–biotin complex cDNA. Unfortunately, I do not have any information concerning the use of this antibody with frozen tissue sections." If you need further assistance, please contact us again.

Thank you for the details that you have provided and I'm sorry to hear that your customer is experiencing difficulty with this antibody. You mentioned that a previously purchased batch worked well; do you have that batch number? Were the protocols and samples the same for both batches? Thank you, and we look forward to hearing from you.