Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody [H5] - ChIP Grade (ab24758)

Hi,
what's the composition and pH of Borate buffer?
Thanks

ANSWER:

Thank you for your inquiry.

The Borate buffer recipe is as follows:

Borate buffer (pH 9.0) 500mL

38.14g boric acid (MW=61.83) to distilled water, make sure all powers dissolve completely.

Adjust pH

Make final volume to 500mL by adding water.

I hope this information is helpful. Please do not hesitate to contact us if you have any additional questions.

can this protocol be applied with magnetic (and not sepharose) beads A or G?

thanks

ANSWER:

Thank you for your reply.

This should be possible. Unfortunately we do not have a specific protocol for this. I would recommend contacting the manufacturer of the beads for more specific information on carrying this out.

I hope this information is helpful. Please do not hesitate to contact us if you have any additional questions.

I recently bought the antibody Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody [H5] - ChIP Grade (ab24758). I wonder which is the best bridging protein IgM-protein A or G to use in ChIP assay.

ANSWER:

Thank you for contacting Abcam regarding ab24758.

Regarding IgM antibodies these do not bind well to either protein A or protein G. We would recommend one of the following alternatives:

1. Protein L beads

2. Couple an IgG anti-IgM antibody to protein A or G beads. I have included the protocol below:

Reagents:
Borate buffer 200 mM + NaCl 3 M pH 9.0
Dimethylpimelimidate (DMP) 20 mM in 200 mM borate buffer + 3 M NaCl, pH 9.0
Ethanolamine 200 mM pH 8 (1:80 dilution of stock ethanolamine)
Phosphate buffered saline (PBS)
PBS + sodium azide 0.1%
Glycine 200 mM pH 2.5
Protein A or G beads (sepharose)

Method:
We recommend using 2 mg of antibody per ml wet beads (use appropriate antibody/protein A or G combination).
- Add 250 mg Protein A or G sepharose beads (beads) to 2 ml PBS + 0.1% sodium azide, for one hour before use. This allows the beads to swell.
- Mix the beads with an appropriate concentration of antibody for 2 hours at room temperature (using a rotator or roller).
- Centrifuge at 2500 rpm for 5 minutes.
- Wash the beads twice in 10X volume of 200 mM borate buffer + 3 M NaCl.
- Resuspend the beads in 20 mM DMP.
- Rotate / agitate the beads for 30 minutes at room temperature.
- Centrifuge the beads at 2500 rpm for 5 minutes.
- Wash the beads 2X in 200 mM borate buffer + 3M NaCl.
- Wash the beads 1X in 20 mM ethanolamine. This stops the coupling reaction.
- Resuspend the beads in 20 mM ethanolamine and rotate for 2 hours at room temperature.
- Centrifuge the beads at 2500 rpm for 5 minutes.
- Wash the beads 2X in PBS.
- Wash the beads 2X in 20 0mM glycine.
- Wash the beads 2X in PBS.

The beads can be stored at 4oC in PBS + 0.1% sodium azide (1 X PBS to 1 X beads)

These beads are now ready for use in immunoprecipitation with IgM antibody.

I hope this information is helpful. Please do not hesitate to contact us if you have any additional questions.

The order number we have for this item is ****. Order was placed 19th or 15th of December 2011



The invoice number is ****if that helps at all?

ANSWER:

Thank you for your reply.

I managed to find the original order and have gone ahead and created a credit note for this product. The credit note number is *****.

Please let me know if there is anything else I can help you with.

The customer would like a credit note for this antibody.



Thank you for your help J

ANSWER:

Thank you for your reply.

I am having difficulty finding the original order, using the PO# ** that you originally sent. I have found an order with the PO#****, the order was placed on**Nov and shipped *** Dec 2011. Is this the correct order? I just want to make sure before I continue with the credit note.

Once I find the correct order, I will go ahead and complete the credit note.

Hope you had a great weekend.