Anti-Histone H3 (acetyl K9) antibody [AH3-120] - ChIP Grade (ab12179)

Thanks for the further information. I have one final question: using the histone extraction protocol, it says to acid extract in 0.2 N HCl; is that 0.2 N added to the extraction buffer, or is it just simply acid diluted in water?

ANSWER:

Thank you for your question.

I hope I can clarify the protocol:

Step 5. is a repetition of step 4.: this provides you the pellet for furtheruse.
Step 6. of the protocol: Re-suspend the pellet in 0.2 N HCl at a density of 4x107 nuclei per ml.



And indeed Step 6. means that you will just use "acid diluted in water" and not the TEB plus 0.2 N HCl.


I have forwarded this to our protocolteam as well and hope they will rephrase it a bit clearer in the future.

Wish you all the best for your experiments.

1) Abcam product code ab1791 and ab12179
2) Abcam order reference number or product batch number ?? ordered in Feb 2012

3) Description of the problem – I don’t know if I’m using the right lysis method to access nuclear proteins
4) Sample preparation:
Type of sample (whole cell lysates, fraction, recombinant protein…): whole cell lysate from HEK 293T
Lysis buffer PBS with 1% Triton-X-100
Protease inhibitors: n/a
Phosphatase inhibitors n/a
Amount of Lysate IPed ug or number of cells did not do an IP; did a western blot of the lysis
Lysate precleared with matrix : yes/no
Positive control
Negative control

5) IP antibody:
Amount/concentration or dilution used
Conjugation:
Antibody noncovalently bound to matrix before incubation with lysate sample?
Antibody noncovalently bound to matrix after incubation with lysate sample?
Antibody covalently bound to matrix - please describe briefly?
Antibody-lysate incubation time:
Incubation temperature

6) IP Matrix (e. beads):
Type of matrix
Amount of matrix

7) Washing after antibody-lysate incubation:
Buffer
Number of washes

8) IP analysis:
Verification method: e.g reprobe in western blotting

Describe verification method briefly:

OR if Western blotting reprobe performed please indicate the following:

Reducing agent DTT
Boiling for ≥5 min? yes
Protein loaded ug/lane or cells/lane .0043 ug

9) Percentage of gel10 %
Type of membrane nitrocellulose
Protein transfer verified yes, using rainbow marker
Blocking agent and concentration .25% nonfat milk in PBST – (used Millipore Snap ID system)
Blocking time 1 minute
Blocking temperature RT

10) Primary antibody (If more than one was used, describe in “additional notes”) : ab1791

Concentration or dilution 2 ug/ml
Diluent buffer PBST + .25% milk
Incubation time 10 minutes (using Millipore Snap ID system)
Incubation temperature: rt

11) Secondary antibody: goat anti-mouse HRP conjugate from pierce
Species:goat
Reacts against: mouse
Concentration or dilution 1:333
Diluent buffer PBST + .25% milk
Incubation time 10 mins (Used Millipore Snap ID system)
Incubation temperature: RT
Fluorochrome or enzyme conjugate: HRP

12) Washing after primary and secondary antibodies:
Buffer PBST
Number of washes 3x30 mls through Millipore Snap ID

Detection method chemiluminescence

Was the method successfully used to detect protein from non-IPed samples? yes

Document attachment: Attaching images of your blot is strongly recommended and can greatly speed up our investigation of your problem.


This questionnaire doesn’t really even seek to answer the basic question I have, which is: should the use of 1% Triton X-100 in my lysis buffer suffice to lyse the nuclei of HEK cells? Or do I need to do something more?

ANSWER:

Thank you for your reply.

I am sorry for the misunderstanding, I assumed that you performed an IP.

As Histones can be a bit tricky, we usually suggest using a nuclear fraction lysate or to follow our Histone extraction protocol (see attachment). In addition, the protein load seems to be a bit low: we usually suggest 20 to 30 ug protein per lane.

Another important step is the gel percentage andmembrane pore size: due to the light nature of this protein, the gel should have a concentration of 15%, and use a nitrocellulose membrane with a pore size of 0.2 μm to ensure optimal capture of Histones proteins.

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

I purchased a couple of your anti-Histone H3 antibodies (ab1791 and ab12179) to use in IPs and Western Blots from HEK cell lysates. I did a trial run of these and also your anti-Sirt6 antibody (ab88494). I saw Sirt6 clearly but could see nothing from either of the anti-H3 at high concentration. I am wondering if possibly I am not lysing the nuclei of the cells with my lysis protocol? Currently i am simply incubating for 1.5 hours at 4 degrees in 1% Triton X-100. If I need to do something more to access the histones, could you please let me know. Thanks!

ANSWER:

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.


I look forward to receiving your reply.

BATCH NUMBER 64800 ORDER NUMBER S 56953

DESCRIPTION OF THE PROBLEM Wrong band size. Detecting a very strong band at 35KDa with low amounts of background, however in your studies AcH3K9 has been detected as a band at 17KDa.

SAMPLE NUclear extract of 3 prostate cell line samples (DU145, LNCaP, and PNT1A)

PRIMARY ANTIBODY Mouse Monoclonal Histone 3 acetylated at lysine 9 2ug/ml(Abcam) diluted in blocking buffer. incubated overnight on an agitator

SECONDARY ANTIBODY Anti-mouse IgG (whole molecule) Alkaline Phosphatase Conjugate

DETECTION METHOD Alkaline phosphatase method

POSITIVE AND NEGATIVE CONTROLS USED none

ANTIBODY STORAGE CONDITIONS 4 degrees C

SAMPLE PREPARATION Nuclear Extraction kit (Pierce) treated with complete protease inhibitor cocktail (Roche)

AMOUNT OF PROTEIN LOADED 30ug

ELECTROPHORESIS/GEL CONDITIONS 4-12% NuPAGE bis/tris gel

TRANSFER AND BLOCKING CONDITIONS Transfer to 0.2um pore size nitrocellulose membrane Block for 2 hours in 3% BSA/TBS Tween

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? boiling samples in reducing agent for 10 minutes instead of 5 minutes running buffer (MOPS to MES) percentage of gel (4-12%) blocking buffer (Marvel to 3% BSA)

ANSWER:

Thank you for your enquiry and I'm sorry to hear that you are experiencing difficulty with ab12179. At this time I would like to make a few suggestions to try to help you out. First, I recommend running a positive control. One recommendation is calf thymus histone prep (such as ab121) - the Abcam lab uses this calf thymus histone preparation to characterize all our chromatin antibodies against histones and histone modifications. For ab12179, this particular antibody was characterized in Western blotting using a whole cell extract of mouse fibroblasts 3T3 cell line treated with sodium butyrate. I have also include our Histone Western blotting protocol which I hope you will find useful. If you have any additional questions, please let me know. Histone Western Blotting This protocol refers to western blot detection of Histone proteins derived from purified calf thymus. Please note that protein loadings derived from cellular lysates will need to be determined empirically. For each gel lane between 1-2 mg of total Histone protein solution in 1x sample buffer should prove sufficient. The choice of sample buffer will vary depending on the type of gel used (i.e. Tris / glycine or Tris / Tricine SDS PAGE). N.B. A higher percentage gel such as 10-20% Tricine SDS-polyacrylamide is recommended for effective resolution of Histone proteins The sample should be supplemented with 5% (v/v) b-mercaptoethanol and heated to around 95°C for 5 min. The sample should be centrifuged briefly to restore sample volume from condensation formation in the eppendorf during heating. Load the protein samples onto the appropriate SDS-polyacrylamide gel and run the gel under standard conditions. NB it is advisable not to run the dye front completely off the gel when dealing with smaller protein resolutions. For protein transfer from the gel please refer to the protocols supplied with your transfer apparatus, as these will vary depending upon the method of transfer employed i.e. semi-dry blotting or wet blotting. N.B. Nitrocellulose membranes with a pore size of 0.2 mm are recommended for optimal retention of Histone proteins. Transfer times between 30 min and 90 min should prove sufficient for effective protein transfer. Visualise equivalent protein loadings using Ponceau staining Block the membranes by adding an appreciable volume of blocking buffer (5% (w/v) BSA, 0.5% (v/v) Tween-20 in TBS or PBS as preferred). Incubate for 1 h at room temperature by gentle rotation. Dilute the primary antibody in blocking buffer as suggested in the datasheet protocols and incubate the blots overnight at 4°C with gentle rotation. N.B. If you wish to perform blocking peptide studies then a final concentration of between 0.1 and 1.0 mg/ml peptide should be pre-incubated with the antibody for around 20 min at room temperature before the blots are added. Rinse the blots briefly in PBS or TBS then perform two 5 min washes in blocking buffer. Prepare the relevant secondary antibody conjugate in blocking buffer and incubate the blots for 1 h at room temperature with gentle rotation. Wash the blots with three 5 min washes in blocking buffer and then perform a final rinse in PBS or TBS. Perform ECL, ECF or infrared detection as described by the manufacturer. Top Tips for Successful Western blotting with our range of Histone antibodies. · Use a high percentage gel for clear resolution of Histone proteins. · Use nitrocellulose membrane with a pore size of 0.2 mm to ensure optimal capture of Histone proteins. · Use high quality BSA in your blocking solutions rather than conventional dried milk such as Marvel.