Anti-CD5 antibody [CRIS1] (Phycoerythrin) (ab1157)

The gels that we have been using are non-reducing, non-denaturing.

The amount of protein loaded vary according to our daily plans.... but should be more than sufficient in order to detect bands. We haven't used loading controls at this point... we wanted to see something first.

We used the NP-40 buffer documented in your western blot beginners pdf file.

In the case of all the antibodies ordered, they have been designated by Abcam as tested for Western blots. Do you have any images on files that I can take a look at? What type of tissue did you use for the testing? How different was your protocol from the one established in our lab? Sorry for all the questions but maybe I can use this info for some of the trouble shooting on our end.

ANSWER:

Thank you for your enquiry.

I appreciate your continued patience in this matter. I have received some feedback from some of the sources of the antibodies that you are enquiring about;

ab7033 - To follow on with the comments that I have made the source of mouse monoclonal [MMP2/2C1] to MMP2 (ab7033) was also concerned with regards the approach that you have used for your sample preparation. I appreciate that you have been using an NP40 buffer extraction. However, this is an approach designed for a cytoplasmic preparation of cell culture cells. I would like to follow up my previous email by recommending that you perform a loading control experiment using an antibody that targets a "housekeeping protein" for example GAPDH or beta actin. This can be performed under the conditions best recognized by the antibody you are using; most likely denaturing, reduced. This will enable you to fully determine the integrity of your protein with respect to its protein composition. My biggest concern with the blot images that you have provided me with are the band doublet that you have been detecting either side of the 37KDa marker as this is present in the majority of your blot images.

The source of ab6586 - Collagen IV antibody (ab6586) makes a similar suggestion although Collagens should in fact be electrophoresed as you have been using non-denaturing, non-reduced conditions. However, it is important that the integrity of your samples are confirmed.

I am still awaiting further information and am in the process of requesting blot images for the antibodies that you have enquired about. I appreciate your continued patience.

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

Hi again.

I submitted the form for ab 2500 (Laminin), however, I am having difficulty re-accessing the questionnaire for the remaining antibodies. For this reason, I will provide the necessary information in this email.

I have not been satisfied with the following antibodies: ab11572, EMMPRIN: (bands not at proper place, band at approx. 37 kDa): I have already submitted images of this. ab7033, MMP2: (bands not at proper place, bands below 40 kDa): I have already submitted images of this. ab6586, Collagen IV: (no bands): I have already submitted images of this. 2 hours transfer period.

Questions regarding: ab3644, MT1-MMP: bands appear at 50 kDa and approximately 40 kDa. MW should be 66 and 54 kDa. What were your results? Images previously sent. ab1828, TIMP2: bands appear at 25 and 37 kDa. Band is documented to appear at 21 kDa. What are your experiences with this? Images previously sent.

For all antibodies: Please find protocol attached to this email. Gels used: Criterion precast (Biorad), non-reducing, percentage determined according to molecular weight investigated All buffers and secondary antibodies used were suggested by Abcam on the data sheets. All protocols included preliminary testing in either 3% skim milk and 3% BSA.

If there is anymore information that I may supply, please do not hesitate to contact me.

Best regards,

ANSWER:

Thank you for your enquiry.

Once again I am sorry to hear of the difficulties that you have experienced using these antibodies. I have had a look through the information that you have provided in conjunction with the blot images and I have a few comments.

Firstly I am concerned about the sample preparation that has been performed. Many of the extraneous bands that have been detected are significantly lower than I would expect. Curiously many are also doublets at approximately 25KDa and 37KDa. My suspicion would be non-specific labeling possibly due to protein degradation. You have highlighted that the samples were prepared as recommended by Abcam. Please can you detail exactly how this was performed as we make many recommendations for sample preparation. My concern with respect to the sample preparation is that many of the proteins that your are targeting are secreted proteins and therefore require delicate sample preparation. Can you tell me whether the skeletal muscle samples were postmortum, biopsies, surgical specimens etc.

I would also appreciate details of the gel conditions that you are using. Similar to my previous response with respect to ab2500 I would appreciate it if you could provide me with details of the gel conditions. I would like to know whether these pre-cast gels are non-denaturing in addition to non-reduced. I would also like details of how the samples are prepared immediately prior to loading on the gel. Can you also provide me with details of the primary and secondary antibody dilutions that have been attempted for each of the samples. Given the non-specific reactivity that you have observed I am also interested whether any no-antibody control experiments have been performed to determine whether the extraneous bands are attricubatble to this reactivity.

I additionally have a few comments with respect to individual antibodies that you have been applying:

In the experiments that have been performed using ab1828, the secondary that has been employed is ab6721. This is an antibody that is specific for use by IHC and has not been tested for use in western blotting.

We have received some excellent feedback as to the application of ab3644 through our Abreviews system. I would like to highlight the importance of a a suitable positive control. For MT1-MMP/MMP14 we recommend the use of placental or breast/lung tissue. For Collagen IV we recommend the use of human epidermal keratinocytes.

I look forward to your comments on these matters.

Please find attached images/comments about our optimization for western blots using your products. We have not been able to produce satisfactory results with the following antibodies: ab2500, ab6586, ab7033, ab1157 We also have questions pertaining to the following antibodies: ab1828, ab3644. We are happy to receive advice from past experiences that Abcam may have had in the process of testing these antibodies and are open to continuing the optimization process. However, if satisfactory results are not obtained, we will count on the Abcam product guarantee.

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with these antibodies. The results that you have been obtaining are certainly troubling. What concerns me the most are the two bands that you are consistently detecting at approximately 25 and 37KDa. You are consistently detecing cross reacting bands and never detecting the endogenous target. Whilst some of the antibodies that you detail have not been tested using rat samples; potentially explaining the absence of reactivity with the endogenous band the presence of the two bands at the aforementioned molecular weight accross many of your samples seems to suggest problems with the secondary antibody. Especially in view of the fact that you have changed this antibody and are in fact using three independent secondary antibodies:

Goat polyclonal to Rabbit IgG H&L (HRP) (ab6721)

Goat polyclonal to Rabbit IgG + Mouse IgG & IgM - prediluted (HRP polymer) (ab2891)

Rabbit polyclonal to Goat IgG H&L (HRP) (ab6741)

I would appreciate it if you could provide me with protocol details of the approach that you are using so that I can better determine potential reasons for these reacting bands. We have not observed such reactivity with any of the antibodies you list. I am particularly interested in the generic protocol that you have been applying. To provide me with these details please click on the link below and complete our on line technical questionaire. If you have modified the protocol for any of the antibodies at any point please mention this. I am also very interested in the method of sample preparation and the control antibody (loading control) that you have used.

http://www.abcam.com/index.html?section=western&pageconfig=technical&intAbID=2500&mode=questionaire

I look forward to hearing from you.