Anti-6X His tag® antibody [4D11] (ab5000)

Hello,
I am very interested in your anti-his antibodies (ab9108, ab18184, ab5000).
However, I was wondering if you could tell me the dissociation constants
of these antibodies to their eppitopes (of any of the ones mentioned
above). It is necessary for me to know for my research.
Kind regards,
--

ANSWER:

Thank you for your inquiry and your interest in our product.

I am sorry to confirm that we do not test / measure the dissociation constants of our antibodies.

I am sorry that I did not have a positive answer for you on this occasion and hope this information is nevertheless helpful. Please do not hesitate to contact us again with any further questions and have a good weekend.

Our customer has the problem with the antibody ab5000 in WB experiment, please check the protocol and the figure .THANK YOU!

1. Order details: a.. Batch number: 57466 b.. Abcam order or Purchase order number: ab5000 c.. Antibody storage conditions (temperature/reconstitution etc) :-20°C

2. Please describe the problem (high background, wrong band size, more bands, no band etc). Many non-specific bands

Lane 1: Sf21 cells infected with AcMNPV

Lane 2: Sf 21 cell lysate

Lane 3: Sf 21 cells infected with AcMNPV-Fve:His6

(Target band was indicated with arrow)

3. On what material are you testing the antibody in WB? · Species: Sf21 insect cells

· Cell extract or Nuclear extract: Cell extract

· Purified protein or Recombinant protein: recombinant protein

3. The lysate a.. How much protein was loaded: 20 ug per lane a.. What lysis buffer was used: PBS containing protease inhibitor (Sigma) and 0.1 % Triton X-1000 b.. What protease inhibitors were used: protease inhibitors cocktails (Sigma) c.. What loading buffer was used: 2x sample buffer d.. Did you heat the samples: Yes e.. temperature and time: 100 °C, 5 mins

4. Electrophoresis/Gel conditions/ Transfer conditions a.. Reducing or non reducing gel: Reducing gel b.. Gel percentage : 15% c.. Transfer conditions: 350 mA

5. Blocking conditions a.. Buffer: TBS b.. Blocking agent: milk, BSA, serum, what percentage: 3 % BSA c.. Incubation time: 1hr d.. Incubation temperature: 25 °C

6. Primary Antibody a.. Specification (in which species was it raised against): mouse mAb · At what dilution(s) have you tested this antibody: 1000X

· What dilution buffer was used: TBS

· Incubation time: 4 h

· Incubation temperature: 25 °C

· What washing steps were done: TBST (TBS containing 0.1 % Tween 20), 1 mins, 3 times

7. Secondary Antibody a.. Specification (in which species was it raised against)? Anti mouse IgG AP-conjugate(Sigma) b.. At what dilution(s) have you tested this antibody: 3000x c.. Incubation time: 2 h d.. Wash steps: TBST (TBS containing 0.1 % Tween 20), 1 mins, 5 times e.. f.. Do you know whether the problems you are experiencing come from the secondary? No

8. Detection method ECl, ECl+, other detection method: NBT/BCIP solution (Sigma)

9. Background bands · Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): no

· Is the blocking step sufficient? yes

· Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) yes

· At what size are the bands migrating? Could they be degradation products of your target?

· Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient)

11. Did you apply positive and negative controls along with the samples? Please specify. 10. Optimization attempts · How many times have you tried the Western?

· Do you obtain the same results every time e.g. are background bands always in the same place?

· What steps have you altered?

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you are experiencing difficulties with this product ab5000 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a replacement/credit note/refund will be offered.

I have looked through the protocols you used and have a few questions and suggestions that might help you resolve the problem.

Can you confirm that the Sf21 cells infected with AcMNPV you used contain an N or C-terminal HHHHHHGS epitope tag?

In most cases, the cause of multiple bands is because the primary and/or secondary antibody concentration is too high and causes non-specific binding. The dilution that we have on the datasheet is a recommended starting dilution and customers are encouraged to determine the optimal concentration/dilution. You can try decreasing the primary (1:2000 ~ 1:5000) and secondary antibody concentration or run a no-primary control (without the primary antibody). Running a no-primary control will be able to eliminate the possibility that your secondary antibody is binding non-specifically. Another possible cause is that the amount of protein is too much. Please try 10ug if you have not already done so.

Please try boiling your protein in SDS-PAGE for 10 minutes rather than 5 minutes to ensure proper disruption of multimers. This will ensure the protein is in the correct conformation to run at the correct molecular weight and be detected by the antibody.

Can you confirm that the second antibody you used also managed to produce good results with other primary antibodies? In some cases, the problem may be with the secondary antibody not binding specifically.

Please try incubating the primary antibody at 4oC overnight. Incubation at this temperature and for longer duration will ensure proper and sufficient binding to the protein of interest.

Washing your blot for at least 15min x 3 times might help decrease non-specific bands.

I hope the above recommendations may already help you. If you still experience problems please do not hesitate to contact me with the answers to the above questions. Also, please advice on how you would to proceed with this enquiry.

I would like to purchase good anti-Histag antibody. Your company sells quite a few anti-Histag. Which one is the most specific for WB (i.e. least cross react in general)? Which one is the most sentive for WB? (i.e. detection limit for detecting least amount of histags). I just put ab5000 as product code because it is monoclonal and would have least cross reactivity.

ANSWER:

Thank you for your enquiry. All our His tag antibodies are of very high quality so it is hard to know which one is best, especially as they have not been tested in parallel.

However, I can tell you that the most popular monoclonal is indeed ab500 and the most popular polyclonal is ab9108.

http://www.abcam.com/index.html?datasheet=500

http://www.abcam.com/index.html?datasheet=9108

I hope this information will be useful,

We have a protein with C- terminal PMAHHHHHH. This ab5000 is able to recognise it? This product recognise any 6-his tag?

ANSWER:

Thank you for your patience.

Following correspondence with Silke Reischl I have determined that this antibody does indeed recognise the classical 6 x His tag HHHHHH, and therefore I would fully expect it to recognise your PMAHHHHHH motif.

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

We have a protein with C- terminal PMAHHHHHH. This ab5000 is able to recognise it? This product recognise any 6-his tag?

ANSWER:

Thank you for your enquiry.

The His tag epitope is HHHHHH. Our Mouse monoclonal [4D11] to 6X His tag (ab5000) was raised using an immunogen against HHHHHHGS. We have had a favourable review left by Silke Reischl, who applied the antibody against N and C terminally tagged his tagged proteins. Unfortunately I do not know whether he tagged the proteins with HHHHHH or a tag that reflects the immunogen sequence above. I have e-mailed Silke in the hope that I will receive an answer that will enable me to determine the likelihood of this antibody recognising your tag sequence: PMAHHHHHH.

I appreciate your patience in this matter and will be in touch once I have an answer.