Anti-SOX9 antibody - ChIP Grade (ab3697)

Dear ,
Here is the reply from customer
Thanks for your reply. I would like you to consider the following statements I have already mentioned.
We tried using SOX9 with our positive controls first. Although the expression of the protein is high in liver and pancreas, human colon tissue normal or inflammed (ulcerative colitis, crohns colitis) have been shown to express this protein very strongly. I can forward a research article on this if needed. We have tried using this antibody on both human and mouse tissue. We have tried this on mouse liver samples we have stored frozen in our freezer. We couldn’t get a staining for any of the experiments.
Regarding fixing, if you can see the report I have tried both acetione and ethanol and that too for 5 and 10 min. I have tried permeablizing as well with both triton x100 and tween 20 in both 0.l% and 0.2%. However, the result is still negative.
The secondary antibodies that we have purchased work fine with beta catenin which is a rabbit monoclonal. Hence, I don’t think there would be any issue with the secondary antibody. My concern is that even if it doesn’t work on paraffin embedded sections, it should atleast work on frozen sections. We have tried all possible things with frozen sections as mentioned in abcam website and additionally we have also tried out our own changes to IHC protocol. We still have the same result – no staining.
Regards

ANSWER:

I appreciate the time taken to submit further information to us.
Reviewing the details, I am sorry there are no further tips to provide on this occasion to help improve the results.
I apologise for the inconvenience and am pleased to offer you either a free of charge replacement for a new vial (unfortunately there are no different lots available), a different anti- SOX9 antibody, or a credit note instead. See below the link to the different alternatives suitable for IHC-P / IHC-Fr:
http://www.abcam.com/index.html?pageconfig=searchresults&search=SOX9&pt=1&sk=app&sv=78&sn=IHC-Fr&l=3&fViewMore=1
Please let me know which of the alternatives offered would be more suitable for you and I will be happy to help.
Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

1) Abcam product code: ab3697

2) Abcam order reference number or product batch number: XXX= XXX

3) Description of the problem:

No Immunostaining

4) Antibody storage conditions (temperature/reconstitution etc):

Received in gel pack, alliquoted and stored in -20 deg C
5) Sample (Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed paraffin embedded sections, cells in culture, other):
Fresh frozen sections and formalin fixed paraffin embedded sections
6) Sample preparation:
cut frozen sections (stored in -80 deg C) warmed for 5 min in RT before starting and feshly cut paraffin embedded sections warmed at 60 deg C before starting the protocol. Blocking:
Yes/No
Yes
Blocking agent and concentration
5% normal goat serum
Blocking time
45 minutes
Blocking temperature
Room temperature
Endogenous peroxidases blocked? Yes/No
Yes
Endogenous biotins blocked? Yes/No
No
8) Fixation Step:
Tried with and without fixation for frozen section, tried acetone and ethanol. No fixing for formalin fixed paraffin sections
Fixation: Yes/No
Fixative agent and concentration
Fixation time
Fixation temperature
9) Antigen retrieval method:Tried Heat induced, microwave heating and enzymatic using trypsin as recommended by Abcam for paraffin sections. Tried with and without antigen retrieval for frozen sections.10) Permeabilization:
Yes/No
Permeabilization agent and concentration
Permeabilization time
Permeabilization temperature
Tried with and without permeablization, tried tween 20 and triton X100. 0.01%, .05% and 1%
Primary antibody (If more than one was used, describe in “additional notes”):
Concentration or dilution 1:25 to 1:250
Diluent buffer
TBS
Incubation time
2 hrs RT, 4 deg C overnight
Incubation temperature
12) Secondary antibody:
Species
Goat
Reacts against
rabbit
Concentration or dilution
1:300 to 1:600
Diluent buffer
TBS or PBS
Incubation time
1 hour RT
Incubation temperature:
Fluorochrome or enzyme conjugate
HRP conjugate
13) Washing:
After primary antibody
After secondary antibody Buffer
Buffer
TBS
Buffer
TBS
Number of washes
3
Number of washes Buffer
3
14) Detection method:
DAB kit invitrogen
15) Positive and negative controls used (please specify):
Negative control is without primary antibody
15) A) How many times you have run this staining?
several
) Do you obtain the same results every time?
No staining achieved so far
C) What steps have you altered to try and optimize the use of this antibody?
I make only one change at a time. I have tried changing time and type of antigen retrieval, tried various dilutions of primary antibody and tried both RT incubation and overnight incubation. Have also tried with and without permeablization
Regards

ANSWER:

Thank you for contacting us.
I am sorry to hear you have been experiencing problems with one of our antibodies. The quality of our products is important to us and I would like to reassure you that we investigate all customer complaints. I appreciate the time taken to submit protocol information and I would like to make some suggestions to the protocol in an attempt to improve the results obtained.
I would also appreciate if you could please send us an image of the tissue.
Unfortunately we have not tested this antibody in paraffin embedded sections, so no tips can be offered; however, I would like to offer some suggestions to improve the staining with frozen sections.
- In cases where no staining is shown, it is crucial to use a positive control in order to find out the source of the problem. I checked the expression of this protein in The Human Protein Atlas. According to it, liver and pancreas human tissue highly express the protein, and therefore are a good suggestion as positive control. Which tissue was used in your experiment? Was it human tissue? Have you check whether the antigen is present in the tissue?
-Regarding fixation, I would recommend using acetone, ethanol or methanol at -20C for no longer than 10 minutes.
-As the antigen is localised in the nucleus, permeabilization may help the antibody reach the epitope. Using NP-40 or Triton (0.1% or 0.2%) in PBS for 10 minutes will partially dissolve the nuclear membrane and therefore will be very suitable for nuclear staining.
-Have you checked whether the secondary works with other primaries? It is crucial to dismiss secondary failure.
-To check whether the substrate has failed, test it by placing a drop of secondary in a slide and adding the substrate.
I hope this information is helpful. Should the suggestions not improve the results, please do not hesitate to contact me again and I will try to provide further help.

Dear Tech Support Team,   Please see below and in the attached file the details of the complaint and the images of the gels.   Kindly advise regarding the appearance of the unspecific bands.   Thanks in advance for your assistance and reply.   Antibody code: AB-3697 Batch number: 288866 Antibody storage conditions (temperature/reconstitution etc): -20. I usually reconstitute this ab in 5% milk' and the problems happen both when it is used fresh and when it is used thawed. Description of the problem (high background, wrong band size, more bands, no band etc.) although according to the data sheet the predicted band size is 65 kD, I get a clear strong band in about 50 kD size, and sometimes almost in 75 kD size. Another problem is that it is not reproducible- some times I see the 65, sometimes the 50 and sometimes the 75 kD band. Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.) human osteoarthritic chondrocytes, cultured in 2D or 3D conditions. Whole cell lysates. Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)10' on ice with  lysis buffer ( 10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.1% NP40, 1 mM PMSF, 1 mM DTT PI roche, 0.001 ug/ml ALLN) 10' on ice with extract  buffer (20 mM HEPES, 0.5 M NaCl, 10 mM KCl, 25% glycerol, 1 mM PMSF, 1 mM DTT PI roche, 0.001 ug/ml ALLN) Amount of protein loaded 15-30 ug Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.) 8-10 % SDS PAGE Transfer and blocking conditions (Buffer/time period, Blocking agent etc.) Tris- Glycin usual transfer buffer (with 30% methanol), blocking 30-60 min  with  5% milk Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) anti sox9, 1-200 in 5% milk, 2 hrs in room. temp.+ over night in rotation in 4ο c Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) sigma anti rabbit IgG (whole molecule) alkaline phosphatase   Detection method (BCIP-NBT reagents, BCIP is an alkaline phosphatase substrate.) Positive and negative controls used (human chondrocytes passage 0-2 without treatment)   Optimization attempts (problem solving)   How many times have you tried the Western? At least 6! Have you run a "No Primary" control? no Do you obtain the same results every time? no What steps have you altered? Time of blocking- between 30-70 min. conc. Of primary ab- 1-2000 To 1:1000. over night- some times I did not incubate, and did only the 2 hrs in Room temp.       Kind Regards,

ANSWER:

Thank you for contacting us. I am sorry to hear your customer is not obtaining the expected results with ab3697.

Having reviewed the protocol there are a few things that I can suggest which may help improve the results currently observed.

Firstly, as the expression of SOX9 seems to be reduced in osteoarthritic chondrocytes it would be beneficial if the positive control suggested on the datasheet (3T3 cells) were tested with the antibody, if your customer has access to this cell type. It appears that with transfection of 293 cells the best results were obtained which may suggest that the expression levels in the OA chondrocytes may be contributing to the problems.

If the 3T3 cells are not available to the customer I would suggest trying to optimise this experiment (the transfected 293 cells) initially then if better results are achieved moving on to optimise experiments with endogenous expression. I would initially not use the PLB buffer as this appears to have detrimental effect on the detection (with comparison of the transfected 293 cell line without PLB).

For these optimisation experiments I would suggest using a higher concentration of the antibody. We suggest using a range of 0.5-2µg/ml for western blotting. This equates to a dilution of 1/400 to 1/100. In addition to blocking with 5% milk, I would also try to block with 5% BSA with 1% BSA in the antibody dilution buffers. With incubation of the primary antibody about for 1 hour at room temperature and overnight at 4°C as your customer has been doing. I would also perform a "no primary" control experiment to ascertain if the non-specific binding of the secondary antibody is producing some of the non-specific bands observed.

I would also suggest reducing the methanol content of the transfer the buffer, we would typically recommend using 20%. If using PVDF membrane this can be reduced further. This may improve the swelling of the gel and transfer of the protein. If these suggestions do not improve the results please let me know.

The antibody is covered by the Abpromise and if satisfactory results are not obtained your customer would be entitled to a free of charge replacement or a credit note.

One of our customer bought #ab3697/Rabbit polyclonal to SOX9 for WB. His sample is fish, he know it was not included in the reactivity of de datasheet. Would you please suggest him some improvement on his application.I have attached the questionnaire for you. Please have a check.Thank you and best regards!

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you are experiencing difficulties with this product ab3697 in WB. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support but please bear in mind that this product has not been tested using fish samples and therefore, might not be suitable for use.

I have looked through the protocols you used and have a few questions and suggestions that might help you resolve the problem. If the suggestions do not prove to be helpful, would you please be so kind to confirm the following items in order to help me better understand the cause of the problem. Most customers who use milk as a blocking agent tend to have problems detecting bands. At Abcam, almost all of our products have been tested with 5% BSA. Sometimes, a different blocking buffer might cause cross reaction between the blocking agent and antibodies, therefore causing weak bands or no bands at all. I would recommend trying 5% BSA, if you have not already done so. Also, cross reaction between the antibodies with the diluent could also cause the lack of signals. We would normally use 1% BSA to dilute the primary and secondary antibodies. You can also use PBST only.

Can you confirm that the second antibody you used also managed to produce good results with other primary antibodies? Was it a donkey anti rabbit IgG antibody? In some cases, the problem may be with the secondary antibody not working.

Please denature your samples for 10 minutes in buffer containing SDS and mercaptoethanol. This will ensure that the protein is in the correct conformation to run at the correct molecular weight and be detected by the antibody.

Could the no signal you are experiencing be due to poor transfer of protein to membrane? Did you check the transfer efficiency? This can be simply done with a reversible stain such as Ponceau S.

If the above suggestions did not help to improve your results, then I am afraid that this product is indeed not suitable for use with fish species.

In any case, can I encourage you to submit an Abreview via the online product datasheet. We always encourage customers to send their results back to us, whether positive or negative, and we make all product information available to other researchers. We reward each Abreview with Abpoints which can be used for discounts off future purchases and gifts. If you decide to submit an Abreview, please include a note that you have already spoken to me, so that I will be able to quickly publish your Abreview, as I have already tried troubleshooting with you.

To find out more about our Abreview system, please see the following URL: http://www.abcam.com/abreviews

Should you require any further information or assistance, please do not hesitate to contact me.

What is the size of the band in Western blot on bovine samples?

ANSWER:

Thank you for your enquiry.

It appears that the datasheet was in error. This antibody was raised to a peptide sequence that is identical in mouse, human, and cow, but has only been tested on mouse tissue. I have updated the datasheet to reflect this. I apologize for the inconvenience.

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