Anti-CD36 antibody [FA6-152] (ab17044)

Thanks for your information about mouse CD36 antibody. I want to detect CD36 from c2c12 cell lines, a mouse cell lines. I just request you if you send me a little aliquot 10 microlitre,I will just check 1 exp, then I will order. I will need a good amount to order. Please let me know it is possible or not.

ANSWER:

Thank you for contacting us. Because we carry over 70,000 products, it isn't feasible for us to keep small sample sizes of our products.

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I hope this information is helpful. Please do not hesitate to contact us again with any other questions.

Have you CD36 mouse antibody to detect endogenous expression. Please let me know.

ANSWER:

Thank you for contacting us.

We have a number of antibodies to CD36 that are guaranteed to detect endogenous expression. For example, we have the mouse monoclonal ab17044 which reacts with human and rat for use in blocking, ICC/IF, WB, IHC-P, IHC-Fr, and Flow Cytometry. We also have the rat monoclonal ab80080 which reacts with mouse and human for use in IHC-P, WB, IP, functional studies, Flow Cytometry, and ICC/IF.

If you could please let me know what species and application you are working in, I would be happy to make more specific recommendations.

Dear technical support:

This customer raised an inquiry about ab17044 (Anti-CD36 antibody [FA6-152]), and this customer used this antibody conduct the western blotassay with human sample, and the data shows the weak signal, because this antibody is already out of our warranty period, therefore she only wants to ask for your kindly help to offer any suggestion to her. I attach the western blot data and the WB questionnaire in this letter, could you please help this customer to solve this problem.

Thanks for your kindly assistance.



Best regards

ANSWER:

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

Having reviewed this case, I would like to address two points of the protocol:

1.) I can suggest to use also BSA as a blocking buffer, as this might help to improve the signal (see also image on datasheet of ab9385).

2.) I can suggest to heat the samples only to 70 degrees Celcius for 10 minutes, as multispan membrane proteins can aggregate if they are boiled.

3.) Can the custoemr use maybe also another cell source as a positive control? Has he used other cells?

Should the suggestions not improve the results, please do let me know, I would be pleased to see how we could help the customer further.

I hope this information is helpful, and I thank you for your cooperation.

Dear Abcam



I have previously used a CD36 antibody from you (ab36977) with success but have noticed that it unfortunately no longer is available. Instead we tried ab78054 (with major problems, which we had a mail correspondence with you about in November 2011) and now we have tried the ab17044 but unfortunately also with problems.



The homogenate used for testing the ab17044 has also been used for the other two antibodies :

Human skeletal muscle tissue homogenized in Tris buffer (25mM Tris, 5mM EDTA, 3% SDS including Protease and phosphatase inhibitors: 20mM β-glycerophosphate, 10mM pyrophosphate,2mM NaOrtovanadate, 2.5mM PMSF, 1 mini comple protease inhibitor tablet (Roche) to 10ml buffer.)

For Antibody ab36977 and ab78054 I also used a homogenate based on the RIPA buffer according to your suggestions. As you can see below in the previous mail, there is basically no difference between the two homogenates, why I only used the Tris-buffer based for this testing as most of our homogenates are based on that buffer.



My problems with the ab17044 is that it gives several bands all very high in the gel. You have previously written to me:

“Regarding the multiple bands with ab36977. This protein undergoes modifications such as glycosylation and palmitoylation which can certainly shift the bands you observe to ˜75 - 80kDa. The ˜50kDa band is likely unmodified protein”

Which make sense, but now the bands are situated even higher in the gel:

I am unsure whether the very intense band above 100kDa are specific?? If I block the signal (by laying something over the membrane when the picture is taken) I get this result, which actually is quite nice, but have I by that removed some CD36 signal? The middle part of the membrane was used for testing another antibody without success.

CD 36 on Lane A-D: all with 15µg total protein.

Lane A:

Blocking for 1½ hour in 5% BSA in TBST at RT

overnight incubation at 4 degrees with antibody diluted in 5% BSA in TBST with agitation (in a tube rolling) with a 1:500 dilution of ab17044.



Lane B:

Blocking for 1½ hour in 5% BSA in TBST at RT

overnight incubation at 4 degrees with antibody diluted in 5% BSA in TBST with agitation (in a tube rolling) with a 1:1000 dilution of ab17044.



Lane C:

Blocking for 1½ hour in 3% BSA in TBST at RT

overnight incubation at 4 degrees with antibody diluted in 3% BSA in TBST with agitation (in a tube rolling) with a 1:500 dilution of ab17044.



Lane D:

Blocking for 1½ hour in 3% BSA in TBST at RT

overnight incubation at 4 degrees with antibody diluted in 3% BSA in TBST with agitation (in a tube rolling) with a 1:1000 dilution of ab17044.



All lanes

Sec. antibody was diluted in 5% BSA in TBST and incubated for 2 hours at 25 degrees in a tube rolling.



All wash steps was in TBST. Both membranes was incubated with ECL and picture was taken with a CCD camera in 1 min.



How many of the bands are specific antibody reactions with CD36 proteins??



Kind regards

ANSWER:

Thank you for taking the time to send us your results. I am sorry to hear you have had difficulty obtaining satisfactory results from this CD36 antibody as well.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

The bands you observe above 100 kDa are likely to be unspecific. Reviewing this case, it seems your sample has not migrated sufficiently into the gel which would also explain that your testing for another antibody failed.

I would like to offer some suggestions to help optimise the results you obtained from ab17044. I would also appreciate if you can confirm some further details:

1) Your target is amembrane protein and it is heavily modified (as discussed before), and these proteins have the unfortunate tendency to aggregate during boiling periods with high temperatures and beta- mercaptoethanol (bME) due to their hydrophobic nature. I would like to suggest to use DTT as reducing agent rather than bME and to decrease the boiling temperature to 65ºC and to prolong the boiling time up to 10 min.

2) What gel percentage have you used and which buffer system? For this target 8-10% gels might be sufficient. Also, the buffer system can have an influence thesize at which a bandruns (see attached).

3) To verify the transfer of the proteins to the membrane I would recommend the Ponceau staining, if you have not already tried (see protocol details attached).

I would hope that these suggestions help to optimise the results and would lead to distinct bands for CD36. It would then not be necessary to 'block out' the unspecific signals (which is also not really common scientific practise). Usually, the specific signal of an antibody is blocked by incubating the antibodywith the immunogenic peptide prior to staining the membrane. This way the specific signal disappears whereas unspecific background staining would still be present, validating the results. Unfortunately, the exact epitope of this particular CD36 antibody is unknown, and therefore, this blocking of the signal is not an option.

I hope this information is helpful for you though, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

both ab17044 and ab64014 did not detect cd36 in pos control red blood cell sample or recombinant cd36 protein. An old antibody, ab36977, did detect cd36 in these samples as expected.

ANSWER:

I am sorry thatthese antibodiesdid not perform as stated on the datasheet. If payment has already been made on the original order and you wish to receive a refund, please ask your purchasing department to contact our accounting department so that we may gather the correct information needed for the refund. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used.

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I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.