Anti-LASP1 antibody (ab1301)

I have tried three of your antibodies with immunohistochemistry but I didn't succeed. I wonder if you have tried them already and in that case which would be the better conditions.

These are the antibodies

PDLIM-1 (ab1301) LASP-1 (ab17022) SM22 alpha (ab10135)

For western blots they don't give me a good signal, there is background maybe due to the secondary goat antibody. Could you please recommend me or send me an specific secondary antibody?

Another question is, I get two bands for LASP-1 around 50 and 35 kDa. Have you seen this before? All my samples are from human tissues and breast cancer cell lines.

Thanks a lot and best wished for the new year!

ANSWER:

Thank you for your email and I'm sorry to hear that you are experiencing some difficulty with these antibodies.

All 3 of these antibodies have been successfully tested for application in Western blotting. However, these antibodies have not yet been tested for application in IHC to our knowledge. They very well may not be suitable for this particular application. I do suggest that you submit Abreviews regarding your experiences with these antibodies in IHC as your feedback is extremely useful for us as well as other customers. To submit an Abreview, just click on the Abreviews tab located on the online product datasheet and then click on the submit an Abreview link. In exchange for an Abreview we will award 50 Abpoints (an additional 100 Abpoints if an image is submitted) which can be exchanged for a variety of awards.

In order to check to see if your secondary antibody is causing the background that you are seeing, you can test this by omitting primary antibody in your protocol and testing with secondary antibody only. If background appears, change either the secondary antibody or the blocking agent. At the bottom of the online datasheets for these antibodies are a list of compatible secondaries (such as ab6741) which are suitable for use with these primary antibodies.

For ab1301 (Goat polyclonal to LASP1), an approximate 30 kDa band was seen in Mouse Brain extracts. I would therefore suggest using this as a positive control.

I hope this helps. Please don't hesitate to contact us again if you need additional assistance with these antibodies. Happy New Year!

I have a question regarding your product ab1301 (LASP-1 antibody). Is this a crude IgG-fraction or an affinity purified antibody?. This is not clear to me, because on the data sheet both is somehow stated:

Purity: IgG fraction

Purification notes: Purified from goat serum by ammonium sulphate precipitation followed by antigen affinity chromatography using the immunizing peptide.

ANSWER:

Thank you for your enquiry. I apologize for the confusion, the antibody is antigen affinity purified: Purified from goat serum by ammonium sulphate precipitation followed by antigen affinity chromatography using the immunizing peptide. If you have any more questions, please contact us again.

I am sorry to hear that no other batch of the antibody is available. I appreciate the refunding and I am happy to help you decide about the suitability of the antibody for others.

The GFP-construct that was used in our lab has been sequenced and contains the full-length human Lasp-1 including the N-terminal region used for the generation of the antibody. Although the GFP protein is fused to the N-terminus of Lasp-1, the antibody could not detect endogenous Lasp-1 in 2 different human cell lines either.

In the datasheet it is stated that Ab1301 will detect human Lasp-1 but the only control that is shown is on mouse brain extract. Therefore, I would recommend to test the antibody on a few human cell lines/tissues before deciding to keep the antibody in your collection or not.

I hope that we will have better luck next time and that my comments are any use to you,

ANSWER:

I have asked our accounts department to refund you for the cost of this antibody. (Order reference 35289).

Thank you for the extra information you have provided about the nature of your fusion constructs. We will follow your advice and have this antibody tested in human cell lines. I have added a note to the datasheet advising potential customers that you have had problems getting this antibody to work.

Your feedback has been most valuable.

In response to our conversation on the phone earlier today about the problems with the Goat-anti-Lasp-1 antibody Ab1301, I hereby give you the details about the conditionns that we used to test the antibody.

Antibody details: Cat. no.: Ab1301-100 Lot. no.: 21380 First time order Aliquoted at 10 ul and stored at -20 degrees Celcius upon arrival.

We used total cell lysates from both HeLa (human cervical carcinoma) and MCF-7 (human mammary adenocarcinoma) cells that were transfected with either GFP alone or GFP-Lasp-1 fusion constructs. These samples were tested in sample preparation buffer containing SDS and beta-mercaptoethanol and were boiled 5 minutes at 95 C. We also split some samples in 2 parts and then one half was not boiled for better antigen conservation.

The extracts were separated on a 4-20% gradient SDS-PAGE gel, transferred to nitrocellulose and stained:

Blocking: 1 hr at RT rocking in 2.5% or 5% skimmed milk in TBS-T (0.05% Tween-20)

Goat-anti-Lasp-1 Ab1301: Overnight at 4 C rocking at 1/500, 1/1000, 1/2500 in 2.5% or 5% milk/TBS-T

Wash: 5 minutes at RT rocking; 3-5 x TBS-T

Rabbit-anti-Goat-HRP (DAKO): 1 hour at RT rocking at 1/1000 and 1/2000 in 2.5% or 5% milk/TBS-T

Wash: 5 minutes at RT rocking; 3-5 x TBS-T

ECL: Pierce ECL reagent 1 minute at RT

Results: 1/1000 and 1/500 dilutions of Ab1301 in 5% milk yielded some background staining and ~20 bands, the strongest of which were 80 and 50 kDa. There was a very faint band at 30 kDa (native Lasp-1) but it was impossible to say whether this was specific given the high number of bands. In GFP-Lasp-1 overexpressing cells no increase in intensity at 58 kDa could be seen compared to empty vector GFP-overexpressing cells.

Blots were stripped and reprobed with Rabbit-anti-GFP Ab290 1/4000 in 1%milk/TBS-T, which resulted in clear GFP (28 kDa) and GFP-Lasp-1 (58kDa) bands.

We also tested the secondary antibody alone (without Ab1301) and this resulted in only a few very faint bands at much higher exposure than in the presence of Ab1301.

Dilution of Ab1301 to 1/2500 and dilution of secondary Ab to 1/2000 and using 2.5% milk/TBS-T reduced the background staining and the number of bands slightly but the intensity of the 30 kDa band also decreased and still no specific 58 kDa band could be observed in GFP-Lasp-1 overexpressing cells. Using unboiled samples only increased the number of bands with Ab1301.

As you can see we have tried a lot of conditions and the antibody still does not work as can be expected since we bought the antibody for detection of endogenous human Lasp-1 and it does not even detect high levels of human Lasp-1 as present in GFP-Lasp-1 overexpressing cells. Therefore, we are asking for a different antibody batch and, if this is not available, for refunding.

I am looking forward to hear from you.

ANSWER:

Your feedback places doubt on the sensitivity/specificity of our antibody and we take it very seriously. As we discussed over the telephone, I will gladly refund you for the cost of this antibody as it clearly has not worked in your research. I'm afraid that we do not have another batch available.

The question now is whether this antibody is suitable to stay in our catalog and for us to continue to sell to other researchers. To this end, your comments on the following questions would be appreciated.

1. Please confirm that the construct you used in the transfection does code for the epitope we used to make the antibody (NPNCARCGKIVYP, from N Terminus of the protein sequence according to NP_006139 - sorry but we have to ask this obvious question) 2. Do you think that the prescence of the GFP tag could be interfering with the binding of the antibody (ie is the tag on the N-terminus?)."

Thank you again for your feedback and I look forward to your response.