Anti-GATA1 antibody - ChIP Grade (ab11963)

please find here below our customer's answers:

- Could you explain the problem? Was this antibody unable to precipitate the chromatin? You have used the previous batch and H3K27ac, how were the results?Yes the new antiboby were not able to precipitate the chromatin whereas the old batch and th Ab against H3K27ac worked very well

- Have you used the same antibody in other applications e.g. WB? No

- We actually incubate chromatin for 4hours at 65C for cross linking reversal. You may try this? We did it

- Could you provide an image?I am sending you a ppt file in attachment.
Kind regards

ANSWER:

Thank you very much for providing images. There are self explanatory.

We are convinced that the vial you received might be faulty or damaged. I am happy to replace the vial. Could you let me know if you would like to try a new lot; the lot we currently have in stock is GR33074.

Could you plase provide the purchase order number?

I will look forward to hearing from you soon.

I have tried to submit the complaint on line, but I can't because we don't have the lot number (customer after aliquoting the Ab has threw off the vial so the lot number is not known).
I'll copy here all the information:

Description of problem: The Antibody did not work for ChIP, compared to an old one bought 2 years ago (same ab from abcam; ab11963; lot number 904877) sample preparation and species: K562(a human cell line) were crosslinked with formaldeyde 1% for 10 minutes. Cells were lysed and the chromatin was prepared by using Branson sonicator.

Experimental conditions: The chromatin from 10 to 25 million cells was incubated with 15ug of the Ab ON.

Controls used: aspecific IgG with the same isotype(negative control); the same Ab ab11963 bought 2 years ago(positive control); Ab against H3K27ac (pos control)

This product has been used successfully in the past (Abcam ab11963)

ChIP

* Catalogue number and lot number (ab11963)

* DNA detection method (regions amplified in PCR, microarray, direct sequencing): qPCR

* DNA purification (decrosslinking conditions, DNA purification method): 65°C ON; QIAGEN PCR purification kit

* Immunoprecipitation conditions (buffer, amount of detergent, primary antibody manufacturer/dilution, incubation time, matrix for isolating antibody complex): see file in attachment

* Sample preparation (X-ChIP/N-ChIP, cross-linking conditions, DNA fragment size): X-ChIP, 10 minutes 1% formaldeyde, 200bp

* Wash steps: see file in attachment

Thanks in advance and kind regards

ANSWER:

Thank you for your email. I am sorry to hear that you have been experiencing problems with this antibody.

I have read the details you have kindly provided and have following further questions for better understanding of the problem;

- What is the purchase order number?
- The ChiP application was added due to the feedback we received from our customer. The feedback is published in the form of Abreview. We unfortunately haven't characterized this antibody in our lab with ChiP so we are unable to provide any ChiP related data.
- Could you explain the problem? Was this antibody unable to precipitate the chromatin? You have used the previous batch and H3K27ac, how were the results?
- Have you used the same antibody in other applications e.g. WB?
- We actually incubate chromatin for 4hours at 65C for cross linking reversal. You may try this?
- Could you provide an image?

I look forward to hearing from you soon.

What is the concentration of this antibody?

ANSWER:

Thank you for your enquiry.

The concentration of this antibody is 1.26 mg/ml.

I hope this information helps. Please do not hesitate to contact us again if you need anything further.

1. Please describe the problem (high background, wrong band size, more bands, no band etc). A lot of unspecific bands and no degradation of my target

2. On what material are you testing the antibody in WB? human cell extract As a positive control we used K-562 cell lysate and as a negative control we used 3T3 lysate.

3.How did you prepare the lysate for the analysis (protease inhibitors etc)? RIPA and Roche complete protease inhibitor

Did you heat the samples? 7 min at 95 C

4. Primary Antibody Rabbit polyclonal to GATA-1 At what dilution(s) have you tested this antibody? 1:2000 Incubation time, wash steps: incubation 1h followed by 3 wash steps ab11963, lot # 86539

5. Secondary Antibody ant-rabbit Ig HRP Amersham At what dilution(s) have you tested this antibody? 1:7500 Incubation time, wash step? incubation 1h followed by 3 wash

Do you know whether the problems you are experiencing come from the secondary? No problems was observed until yet

6. What detection method are you using? ECL Amersham

No problems was observed until yet for the secondary antibody for the same cell lysates with other primary antibodies

Is the blocking step sufficient? Blocking was performed in 5% non-fat dray milk over night at 4C.

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? A lot of unspecific bands and no degradation of my target

8. Optimization attempts How many times have you tried the Western? two Do you obtain the same results every time? yes

What steps have you altered? 4 washing steps

9. Did you apply positive and negative controls along with the samples? Please specify. Yes. As a positive control we used K-562 cell lysate and as a negative control we used 3T3 lysate.

ANSWER:

We have just received the following information from the source of ab11963:

"you can get some unspecific bands with this antibody but it will yield a much stronger signal for GATA-1 at 47 KDa. I should encourage the customer to try the antibody more diluted (1:5000 and 1:10000) and see how it goes."

I hope this advice helps, if you still are not satisfied with this product please let me know and we will arrange a refund.

BATCH NUMBER 86539 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM Abcam Troubleshooting form ? Western Blot-

1. Please describe the problem (high background, wrong band size, more bands, no band etc).

2. On what material are you testing the antibody in WB? ? Species? human ? Cell extract/ Nuclear extract? Cell extract ? Purified protein? no ? Recombinant protein? no

3. How much protein did you load? ? How did you prepare the lysate for the analysis (protease inhibitors etc)?

RIPA and Roche ?complete? protease inhibitor

? Did you heat the samples?

7 min at 95? C

4. Primary Antibody ? Specification (in which species was it raised against)? Rabbit polyclonal to GATA-1 ? At what dilution(s) have you tested this antibody? 1:2000 ? Incubation time, wash step? incubation 1h followed by 3 wash steps

ab11963, lot # 86539

5. Secondary Antibody ? Specification (in which species was it raised against)? ant-rabbit Ig HRP Amersham ? At what dilution(s) have you tested this antibody? 1:7500 ? Incubation time, wash step? incubation 1h followed by 3 wash ? Do you know whether the problems you are experiencing come from the secondary? No problems was observed until yet

6. What detection method are you using? ECL Amersham

7. Background bands ? Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a ?No primary? control) No problems was observed until yet for the secondary antibody for the same cell lysates with other primary antibodies

? Is the blocking step sufficient? (We recommend blocking the membrane by adding 20 ml of blocking buffer (5% non-fat dry milk, 0.1% Tween-20 in TBS). Incubate for 2 h at room temperature or overnight at 4?C with agitation) in combination with other antibodies no background was observed Blocking was performed in 5% non-fat dray milk over night at 4? C.

? 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? A lot of unspecific bands and no degradation of my target

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

8. Optimization attempts ? How many times have you tried the Western? two

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

? What steps have you altered?

4 washing steps

9. Did you apply positive and negative controls along with the samples? Please specify. Yes. As a positive control we used K-562 cell lysate and as a negative control we used 3T3 lysate.

ADDITIONAL NOTES Thanks in advance mit freundlichen Gruessen/Best Regards BIOZOL Diagnostica Vertrieb GmbH Dr. Ralf B?uerle

von/from Dr. Ralf Baeuerle BIOZOL Diagnostica Vertrieb GmbH Postfach/P.O. Box 2022 D-85386 Eching Germany

Fon +49 (0)89 3799 666-6 Fax +49 (0)89 3799 666-99

e-mail r.baeuerle@biozol.com oder/or r.baeuerle@biozol.de Website http://www.biozol.com oder/or http://www.biozol.de

ANSWER:

I'm sorry to hear you are having a problem with ab11963. I enclose the recommended protocol for this antibody and would recommend extensive washes and the addition of 0.1% Tween 20 to all solutions as this will significantly increase the antibody specificity and decrease the background.

Electrophoresis 1. Nuclear extracts are prepared according to Dignam's procedure and freeze aliquoted on liquid nitrogen. Prior to electrophoresis, samples are thawed on ice and diluted in 4X sample buffer (250 mM Tris-HCl, pH 6.8, 8% SDS, 40% glycerol, 20% 2-mercaptoethanol, 2% bromophenol blue). Samples are then boiled 3-5 min., spun down, loaded onto a 4-20% SDS-PAGE gel and run until migration front reaches the bottom of the gel. We suggest that 20 µg of the recommended positive control be loaded into the appropriate well.

Transfer 2. Transfer protein to polyvinylidene difluoride (PVDF) membranes at 400 mA for 90 minutes using the buffer described by Towbin et al2 (25 mM Tris, 192 mM glycine, 15% methanol).

Blocking 3. Block membranes by incubating overnight at 4°C with shaking in 5% Blotto (5% dry, non-fat skim milk powder in PBS (pH 7.4)).

Incubation with Primary Antibody: For 4 cm2 membranes, we recommend using a final volume of 1 ml of diluted antibody solution. To obtain comparable results for a 20 cm2 membrane, use 5 mls of the diluted antibody solution.

4. Rinse membranes with 5% Blotto. Dilute the antibody (at the appropriate dilution) in 5% Blotto. Incubate for 1 hour at 37°C, 2 hours at RT (room temperature) or overnight at 4°C with agitation.

5. Decant antibody solution. Wash the membrane 5 times for 5-10 minutes at RT in 1% Blotto (1% dry, non-fat skim milk powder in PBS (pH 7.4)). Washing should be performed with vigorous agitation over a minimum 30-minute period.

Incubation with Secondary Antibody 6. Incubate membranes with secondary HRP conjugate diluted in 5% Blotto for 1 hour at RT with gentle shaking. The dilution of the secondary antibody conjugate will vary according to manufacturer's specifications; however, we recommend using a 1:2000-1:5000 dilution of conjugated antibody. 7. Repeat step 5. 8. Wash membrane with PBS (pH 7.4) for 5 minutes with agitation before proceeding to the chemiluminescence reaction.

Chemiluminescence Reaction 8. Prepare and use the chemiluminescent substrate according to the manufacturer's instructions. 9. Immediately wrap membrane and expose to X-ray film for 15 seconds to a 1-hour period. The time exposure may vary according to the amount of antigen and should be optimized for each source. Please note that the background can increase with time exposure.

Reference 1. Dignam, J.D., et al., (1983), Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei, Nucleic Acids Res., 11(5), 1475-1489.

Please let me know if this helps and do not hesitate to contact us with an image for further advice if you still have a problem,