Goat polyclonal Secondary Antibody to Guinea pig IgG - H&L (TR) (ab6906)

Thank you so much for your reply. Yes, I did washing with 1xPBS 3 times after fixing, after permeablization and after primary antibody incubation. If I can, I'd like to replace this product with product ab7137 which is pre-absorbed so that the chances to get background may be lower.

ANSWER:

I have arranged a free replacement vial of ab7137 to be sent to you on order 161903 today. I hope you will be satisfied with this product.

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER ER52866

DESCRIPTION OF THE PROBLEM high background even without primary antibody

SAMPLE human epidermal cells: keratinocytes

PRIMARY ANTIBODY ab7842: Guinea pig polyclonal to insulin

DETECTION METHOD Nikon microscope

ANTIBODY STORAGE CONDITIONS just received the day before, and store at 4 centigrade

FIXATION OF SAMPLE 4% paraformaldehyde 15 minutes

ANTIGEN RETRIEVAL No

PERMEABILIZATION STEP 0.25% Triton-x100 10minutes

BLOCKING CONDITIONS 1%BSA in PBS for 30minutes

SECONDARY ANTIBODY ab6906: Goat polyclonal to Guinea Pig IgG H&L (texas red)

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? I tried different dilution rate: 1:200, 1:1000, 1:2000,

I also tried different block solution, 1%BSA, 5%BSA, 1%milk, 5%milk, and 1%BSA with 5% goat serum

ANSWER:

I'm very sorry to hear you are having problems with ab6906. The fact that a no primary test gave you high background is very concerning and it certainly suggests the vial you received was damaged during shipping or storage. I looked at the tracking information of your order and it seemed that the package was delayed as fedex have recorded "Customer not available or business closed" and delivered the parcel at another address first. Can I please verify that you wash in PBS several times in between steps to remove the paraformaldehyde and primary antibody?

Can you please tell if if you would like a refund or a replacement?

I look forward to hearing from you,

Is this antibody tested for reactivity against other species IgG as ab7137? Sincerely

ANSWER:

Thank you for your enquiry.

This antibody has not been preabsorbed against other species IgG.

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

I have a question about the conjugation method used to conjugate the Texas Red to the antibodies. I'm doing a secondary conjugation and for some reason they only want to attach to the antibodies attached to the Texas Red, so I'd like to know the linker used.

ANSWER:

The Texas Red we use for the conjugation is Texas Red® sulfonyl chloride. The active functional group of the dye reacts with a primary amine of the antibody and forms a covalent bond. If the secondary conjugation is targeting the amine or carboxyl or sulfhydral groups, I am not surprised that it only wants to attach to the antibody.

I hope this information helps. If you have any additional questions, please contact us again.