Anti-Histone H3 (citrulline 2 + 8 + 17) antibody - ChIP Grade (ab5103)

send indirect ELISA protocol

ANSWER:

Thank you for contacting us.

The link to our indirect ELISA protocol is:
http://www.abcam.com/index.html?pageconfig=resource&rid=11389

I have also attached the protocol as pdf-file.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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I would like to test ab6464 anti-citrulline and ab5103 anti-Histone H3 Citrulline on FFPE mouse tissue for IHC. Thanks!

ANSWER:

Thank you for your email.

DISCOUNT CODE: xxx for ab6464
DISCOUNT CODE: xxx for ab5103
Expiration date: xxx

I am very pleased to hear you would like to accept our offer and test ab6464 and ab5103in IHC-P. Each code will give you: 1 free PRIMARY ANTIBODY before the expiration date. To redeem this offer, please submit an Abreview for IHC-Pand include this code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active.
For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: http://www.abcam.com/collaborationdiscount.

Order Details
Antibody code:ab5103

Problem
Choose:No signal

Lot number:GR51273-1

General Information
Antibody storage conditions (temperature/reconstitution etc):Aliquoted out upon arrival and stored at -20 degrees celcius until used


Description of the problem (high background, wrong band size, more bands, no band etc.):No bands


Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.):Commercial HL60 whole cell lysate (abcam ab7914 Lot GR21162-1)


Sample preparation (Buffer/Protease inhibitors/Heating sample etc.):No sample prep needed, according to product sheet, HL60 whole cell lysate comes pre-reduced


Amount of protein loaded:20ug (20ul of 1ug/ul)


Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.):10% reducing gel, ˜60 minute running at 200V, fully submerged electrophoresis


Transfer and blocking conditions (Buffer/time period, Blocking agent etc.):Transfer for 120minutes at 100V in transfer buffer with an ice block, blocked for both 60 minutes and overnight (separate membranes) with 5% skimmed milk powder reconstituted with PBS + 0.1% Tween 20


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step):Abcam ab5103 anti-Cit[H3], rabbit polyclonal antibody, in5% skimmed milk powder reconstituted with PBS + 0.1% Tween 20, at a concentration of 1:1000 for both 60 minutes and overnight (separate membranes). Washed 3 times for 10 minutes each inPBS + 0.1% Tween 20


Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step):Sigma A0545 HRP conjugated anti-rabbit, raised in goat,in5% skimmed milk powder reconstituted with PBS + 0.1% Tween 20, at a concentration of1:5000 for 60 minutes.Washed 3 times for 10 minutes each inPBS + 0.1% Tween 20


Detection method (ECL, ECLPlus etc.):


Positive and negative controls used (please specify):Positive: Abcam ab7914 HL60 whole cell lysate, no negative control



Optimization attempts (problem solving)
How many times have you tried the Western?Three times, twice by me and a third western was carried out by a lab technician to eliminate handling issues

Have you run a "No Primary" control?
Yes

Do you obtain the same results every time?
Yes

What steps have you altered?
The length of the block and primary antibody incubations

Additional Notes:


Image:
I have attached images of a coomassie stained gel that wasn't transferred, a coomassie stained gel that was transferred, the transfer membrane probed with ponceau red and the transfer membrane probed with antibodies. In all cases M1 is bio-rad precision plus marker and M2 is invitrogen magic mark XP.

ANSWER:

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will provide us with vital information for our monitoring of product quality. I would like to reassure you that this antibody is tested and covered by our guarantee for ab5103 and human samples.

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. Reviewing the details, I am sorry there very few tips to provide on this occasion to help improve the results. However, I can recommend you may like to consider trying the following options:

1. I can confirm the lysate contains reducing and denaturing agents, but will need to be heated to 95oC for 5 - 10 minutes in order to fully denature.

2. I can recommend to try the antibody at a higher concentration to help improve the signal, try 1:500. Dilutions provided on the datasheet are a recommendation only and we suggest these may require individual optimization.

Alternatively, or if these tips do not work, I am pleased to offer you a free of charge replacement or credit note in compensation for this antibodyifis hasbeen purchased in the last 6 months. I am sorry, we have a few order from Ireland for this item, and regrettably I am not able to trace the order number provided.I would appreciate if you are able to provide the Abcam order reference number and date of purchase for this vial?

Thank you for your cooperation. I look forward to hearing from you with the results and/or details of how you would like to proceed.

No staining with ab5103 in WB, human samples
used ab7914 as a positive control lysate
We've used 2vials unsuccesfully.

ANSWER:

Thank you for your telephone call yesterday afternoon. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee for WB and human samples. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

As discussed on the telephone, I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily. If you have ordered twice, I would appreciate if you could confirm both the order numbers on the form.

I would appreciate if you could also provide an image which would help us to assess the results.

Thank you for your time and cooperation. We look forward to receiving the completed questionnaire.


Order Details
Antibody code:


Problem
Choose: Non-specific band Multiple bands No signal or weak signal High background

Lot number

Purchase order number
or preferably Abcam order number:



General Information
Antibody storage conditions (temperature/reconstitution etc)


Description of the problem (high background, wrong band size, more bands, no band etc.)


Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)


Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)


Amount of protein loaded


Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)


Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method (ECL, ECLPlus etc.)


Positive and negative controls used (please specify)



Optimization attempts (problem solving)
How many times have you tried the Western?



Have you run a "No Primary" control?
Yes No

Do you obtain the same results every time?
Yes No
e.g. are the background bands always in the same place?


What steps have you altered?


Additional Notes:


Image:
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.

I have tested mouse bronchoalveolar lavage fluid and mouse neutrophil lysates with single bands seen in these samples. I did not run any controls on this blot and have not tried blocking with BSA. Attached is a copy with the blot with the first and last lane being molecular ladders representing 22, 16 and 6 kDa.

ANSWER:

Thank you for your reply.

Due to the various other modifications to this protein, it is likely that the shifted bands represent modified Histone H3 that are present in this sample, but not your other samples. Such bands are also observed in our own validation WB and can be blocked with the peptide ab32876.


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