Anti-Transferrin Receptor antibody [B349 (DF1513)] (ab8598)

Would these products
(ab1086, ab84036, ab9179, ab8598) detect Trf1 and Trf2?

ANSWER:

Thank you for your enquiry.

We have not tested these products (ab1086, ab84036, ab9179) for cross reactivity with Trf2. However, based on immunogen sequence (ClustalW analysis, Score 23.0), we do not expect to see any cross reactivity whatsoever. However, there might be a possibility that ab8598 could react but we do not supporting data.

I hope this information will help settle any concerns that you may have.

I recently ordered the Anti-Transferrin Receptor antibody [B349 (DF1513)] (ab8598) from you. I have used it at 1:100, in 1% Milk TBST at 4 oC overnight followed by a secondary anti mouse HRP antibody 1:3000. I got no signal from the antidody, a few weak bands but nothing that I think was specific. Are there any specific conditions that this antibody should be usedunderso I canget it working a little better? Any information will be very much appreciated.

ANSWER:

Thank you for your inquiry.


We have confirmedTFR expression in Hep G2, HEL 92.1.7 and TF-1 whole cell lysates using thefollowing conditions:

Load 50 ug of total protein
Blocking with 5% milk TBST for one hour at room temperature
Primary: 1:100 incubate for one hour at room temperature with 5% milk TBST
Secondary: in-house secondarygoat anti-mouse IgG-HRP: at 1:3000. Incubate for 45 minutes
10% gelPVDF membranes

If the above does not help, I can help if you provide more detail on the protocol:

1. What samples are you testing?
2. How much protein were you loading?
3. What did you do for the blocking step?
4. Have you confirmed the secondary has been performing?
5. Did you run a loading control?
6. Did you load a positive control?

I hope this information helps. Please contact us with any other questions.

I spoke to you today about the Anti-transferrin receptor antibody (ab1086) that did not work for my cell line (Jurkat). I would like to thank you for offering to send us a replacement.  I would be interested in trying the western blot again with ab8598

(the antibody that you recommended, which seems to have worked quite well with a number of cell lines).

Please let me know if you need any further information from me.

ANSWER:

Thanks for following up with me.

I would be happy to send you a vial of ab8598. I will just need to locate your initial order for ab1086 to process your request. Do you have the purchase order number or Abcam order reference number associated with the purchase of ab1086?

Thanks!

BATCH NUMBER 128772 ORDER NUMBER ?

DESCRIPTION OF THE PROBLEM No signal or weak signal

SAMPLE cell lysate of Caco-2 cells

PRIMARY ANTIBODY your anti-TfR antibody, catalog number ab8598, lot 128772, dilution 1:100 = 4 ug/mL. 2 hours room temp, washing etc with TBS/Tween; blocking beforehand with 5% milk in TBS/Tween

DETECTION METHOD ECL from Perkin Elmer

POSITIVE AND NEGATIVE CONTROLS USED We thought the Caco-2 lysate was going to be our positive control for our subsequent experiments.

ANTIBODY STORAGE CONDITIONS 4 degrees

SAMPLE PREPARATION cells in a 6 well plate lysed in 100 uL Laemmli sample buffer with 10 mM DTT and protease inhibitor cocktail set V at the recommended concentration. AFter addition of sample buffer, cells were scraped from the bottom with the back of a pipet tip, pipetted up and down in an insulin syringe to make it less viscous, boiled for 10 min at 99 degrees and stored at -20 overnight.

AMOUNT OF PROTEIN LOADED 20 uL of the above solution

ELECTROPHORESIS/GEL CONDITIONS 10% SDS gel

TRANSFER AND BLOCKING CONDITIONS transfer buffer is tris/glycine with methanol, 450 mAmp-hour (45 mAmp for 10 hours in cold room).

SECONDARY ANTIBODY Goat anti mouse-HRP, [another company], 1:1000 dilution in blocking buffer

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No

ADDITIONAL NOTES Does this antibody only recognize TfR in its native form perhaps? Since in the application notes it sais it can only be used on frozen sections, I'm starting to think that... Please let me know any tips you might have.

Thanks.

ANSWER:

Further to contact with the source of this antibody I have determined that this antibody does not perform well by western blotting. Given that this antibody was detailed as working by western blotting on our datasheets I would like to firstly apolagise and secondly offer you a credit note to the value of one vial of this antibody.

Please e-mail me details of your original purchase including the order number and date of purchase and I will arrange for our accounts team to raise you the credit.

BATCH NUMBER 128772 ORDER NUMBER ?

DESCRIPTION OF THE PROBLEM No signal or weak signal

SAMPLE cell lysate of Caco-2 cells

PRIMARY ANTIBODY your anti-TfR antibody, catalog number ab8598, lot 128772, dilution 1:100 = 4 ug/mL. 2 hours room temp, washing etc with TBS/Tween; blocking beforehand with 5% milk in TBS/Tween

DETECTION METHOD ECL from Perkin Elmer

POSITIVE AND NEGATIVE CONTROLS USED We thought the Caco-2 lysate was going to be our positive control for our subsequent experiments.

ANTIBODY STORAGE CONDITIONS 4 degrees

SAMPLE PREPARATION cells in a 6 well plate lysed in 100 uL Laemmli sample buffer with 10 mM DTT and protease inhibitor cocktail set V at the recommended concentration. AFter addition of sample buffer, cells were scraped from the bottom with the back of a pipet tip, pipetted up and down in an insulin syringe to make it less viscous, boiled for 10 min at 99 degrees and stored at -20 overnight.

AMOUNT OF PROTEIN LOADED 20 uL of the above solution

ELECTROPHORESIS/GEL CONDITIONS 10% SDS gel

TRANSFER AND BLOCKING CONDITIONS transfer buffer is tris/glycine with methanol, 450 mAmp-hour (45 mAmp for 10 hours in cold room).

SECONDARY ANTIBODY Goat anti mouse-HRP, [another company], 1:1000 dilution in blocking buffer

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No

ADDITIONAL NOTES Does this antibody only recognize TfR in its native form perhaps? Since in the application notes it sais it can only be used on frozen sections, I'm starting to think that... Please let me know any tips you might have.

Thanks.

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with this antibody. I have read your technical questionnaire and I have a few comments.

Whilst we recommend that our customers use a tonsil preparation as a positive control I have performed a brief search of the literature and it appears that the use of Caco-2 cells is quite standard for the studying of Transferrin Receptor expression.

I would like to recommend that you quantitate the mass of protein that you are loading onto the gel; presently there is no way of determining the mass of protein loaded per well. This will provide a better idea of whether the antibody is capable of detecting its target by immunoblotting. It would also help if you ponceau stain your membrane following transfer. This will enable you to determine the integrity of the protein and the success of transfer. Alternatively a parallel loading control western blot would serve to confirm the integrity and transfer of your samples.

I will submit your question regarding the antibody only targeting the native form of this protein to the lab and get back to you shortly. I appreciate your patience.