Anti-CXCR4 (phospho S339) antibody (ab74012)

Dear ,

Please find the completed sheet.
1) Abcam product code ab
ab74012
2) Abcam order reference number or product batch number
Lot:GR51025-1
3) Description of the problem
No specific band was detected
4) Sample preparation:
Type of sample (whole cell lysates, fraction, recombinant protein…): whole cell lysate (treated with SDF-1 or pervanadate vs untreated, cells have been starved or not starved)
Lysis buffer : RIPA
50 mM TrisHCl pH7.4; 150 mM NaCl; 2 mM EDTA; 1% NP-40; 0.1%SDS
1 liter:
1 M tris 7.4 (invitrogen #15567-027) 50 ml
5M NaCl 30 ml
0.5M EDTA (Gibco #15575-038) 4 ml
NP40 10 ml
10%SDS (Bio-rad #161-0416) 10 ml
Add the following inhibitors just prior use:
PIC (protease inhibitor cocktail):100x Sigma #P8340
DTT 1M : 1000X (1mM final) Sigma #646563
NaF 0.5M: 100X (5mM final) Fluka # 67414
Na3VO4 200mM: 100X(2mM final) Sigma #S6508
Protease inhibitors: see above
Phosphatase inhibitors : see above
Reducing agent: see above
Boiling for ≥5 min? yes and no (95°C and 37°C)
Protein loaded ug/lane or cells/lane: 10µg
Positive control : 100nM SDF-1, 50µM pervadate
Negative control : water
5) Percentage of gel
Type of membrane : Immobilon-P transfer membrane, 0.45µm Millipore #IPVH00010
Protein transfer verified: yes
Blocking agent and concentration: milk or BSA, 5% each in PBST or TBST
Blocking time : 2h at 4°C
Blocking temperature: 2h at 4°C
6) Was a fresh membrane used or had it been probed and stripped with a different antibody?
Fresh membrane used
7) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution : 1:500
Diluent buffer: 5%BSA/PBST, 5%BSA/TBST, 5%milk/PBST, 5%milk/TBST
Incubation time: overnight at 4°C
Incubation temperature: overnight at 4°C
8) Secondary antibody:
Species: donkey anti-rabbit HRP Jackson Immuno Research #705-035-152
Reacts against:
Concentration or dilution : 1:5000
Diluent buffer: 5%BSA/PBST, 5%BSA/TBST, 5%milk/PBST, 5%milk/TBST
Incubation time 30min at 4°C
Incubation temperature: 30min at 4°C
Fluorochrome or enzyme conjugate: HRP
9) Washing after primary and secondary antibodies:
Buffer: PBST or TBST
Number of washes: 3 times
10)Detection method: chemiluminescence, Immobilon Western Millipore #WBKLS0500
11) How many times have you run this staining? 3 times
Do you obtain the same results every time? yes
What steps have you altered to try and optimize the use of this antibody? Blocking and buffer, heating or none heating of samples before loading
blocked with 5%BSA/PBS
blocked with 5%milk/PBST
Thanks for your answer.
Best Regards

ANSWER:

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab74012. I would also appreciate if you can confirm some further details:

What cell line was used for the western blot? And for how long were they treated with the SDF-1 and pervadate?

The Western blot presented on the datasheet of this antibody was performed using a 1/500 dilution with overnight incubation at 4°Cas you have been doing, however a larger amount of protein was loaded,60 ug. BSA was used for blocking.

I would suggest rying the following:

1. Heating the protein sample to 70°C for 10 minutes prior to loading onto the gel. As the protein is a multi-pass membrane protein heating to 95°C can sometimes lead to aggregation.

2. I would suggest loading more protein onto the gel. Initially try 30 ug per lane.

3. I would block the membrane using 3% BSA instead of milk as milk contains the phosphoprotein casein which can sometimes increase the background observed. I would also increase thetemperature of the blocking step to room temperature for 2 hours.

4. I would incubate the secondary antibody for a little longer, 2 hours at room temperature.

Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

Hi,

I have tested your antibody in a cell line expressing CXCR4, with BSA or milk as blocking reagent, in PBST or TBST, but I could not detect a specific band. Please could you provide me with the exact protocol.

Thanks for your help

Best Regards

ANSWER:

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with the anti-CXCR4 (phospho S339) antibody (ab74012). We take product complaints very seriously, and investigate every product that we feel may not be performing correctly. I have contacted the source of this antibody to obtain the exact protocol used to produce the Western blot image presented on the datasheet of ab74012.

In the meantime, I may be able to help in optimising the protocol if you wouldn't mind sharing it with me? This will also allow me to investigate this problem further and initiate any additional testing where necessary. To this end I am attachinga questionnaire. If you could also include an image of the results obtained that would be very helpful to further understand the problems encountered.

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.

Thank you for the suggestions, which I think are quite useful. I'm quite sure that the antibody detect the phosphorylated form of CXCR4 rather than unphosphorylated one but the point is whether it also binds to other protein containing the same epitope since it's so short? In my case, I used CXCR4 null section which does not contain CXCR4 protein thus there shouldn't be any staining. But I observed quite strong puncta-like staining in nucleus, not all nuclei. So I blast the epitope, as I emailed you, there are lots of hits with 100% match with the immunogen sequence. I think, according to the datasheet, the immunogen is HSSpVS, but I'm not very sure about it. Can you confirm this and I will not be bothered with this uncertainty. Thanks!

ANSWER:

Thank you for your response and for providing some further details.

I would like to clarify a few points in this e-mail.

This (ab74012: Anti-CXCR4 (phospho S339)) is a polyclonal antibody (not a monoclonal) and will therefore consist of a mixed population of IgGs each of which will recognize a different epitope. For polyclonal antibodies, the epitope sequence is not mapped. Such studies can only be carried out for monoclonal antibodies. The epitope of ab74012 is unknown since it has not been determined.

The exact sequence of the immunogen used to raise this antibody is commercially sensitive information so it is not provided on the on-line datasheet. It was a synthetic phosphopeptide derived from human CXCR4 around the phosphorylation site of serine 339 (H-S-SP-V-S).

The specificity of this antibody is QC-d on a regular base.

1. The immunogen peptide must be HPLC purified and MS to confirm the sequence is correct.

2. The antibody binding can be blocked specifically with the immunogen peptide.

3. For phospho-specific antibody, ELISA data must show that this antibody react with phosphor-peptide, not the counterpart (non-phospho-peptide with the same amino acid sequence).

I have searched the immunogen sequence using the database from UniProt and didn’t find any significant homology with other proteins. Could you please specify and confirm what blast website you used and what results were.

All our customer feedback, including complaints are monitored weekly by our in house technical support team. If a product is at fault the technical support team will consider removing the product from our catalogue in order to avoid future customer inconvenience. We have been selling this antibody since 2009. According to our record, this is the first complaint we have received so not aware of any specificity issues.

As recommended in the previous e-mail, blocking phospho protein properly is essential. For detection of phospho protein, we would suggest using 1-3% BSA solution - as blocking. Some other types of blocking agents may lead to significant decrease or loss of signal intensity due to reaction of primary antibody with blocking/diluting agent.

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

Thank you for the reply. I haven't taken any images since it seemed useless for me but I will. Regarding to your concerns: Sample: embryonic brains were dissected out and put in 4% PFA o/n at 4C. Cryostat sections were cut at 14um thick. Stored in -20C until use. Blocking: 10% normal donkey serum in PBS45 mins at RT Positive control: I have sections treated with protein phosphotase which showed no signal. This means the antibody did recognize the phosphorylated protein. But the thing is since the immunising peptide is so short that it is inevitable to cross reactive for other proteins. I wonder if you have blasted the sequence. If you do so, you'll see how many hits will come out, some of which are localized in nucleus. Secondary control: the secondary antibody worked fine because I had other sections done at the same experiment and they didn't show the same nuclear staining. And I have done the control experiment with the same secondary antibody in other experiment. All in all, I just want to make sure the immunising peptide's sequence is what is stated in the datasheet, ie,HSSpVS. If so, the cross reactivation is inevitable. I look forward to your comments. Thanks!

ANSWER:

Thank you for getting back to me and for providing some further details.

This antibody detects endogenous levels of CXCR4 only when phosphorylated at serine 339 (Mouse: Ser 346).

After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:

1) Do you know if the samples contain phosphorylated CXCR4? Have you had any chance to use freshly fixed samples?

2) For detection of phospho protein, we would suggest using 1-3% BSA solution - as blocking. Some other types of blocking agents may lead to significant decrease or loss of signal intensity due to reaction of primary antibody with blocking/diluting agent.

I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.

Product code: 74012
Lot number: 938616
Inquiry: Blocking: 10% donkey serum in PBS Primary Ab: 1:100, 1 hr at room temperature Fixation: 4%PFA, overnight Secondary: secondary antibody conjugated with Alexa 488 1:1000 45mins at room temperature wash: 3x5 min in PBS (0.1% TritonX-100) Problem: I used CXCR4-/- mouse embryonic brain section as control and found nuclear staining. I blast the sequence of immunising phosphopeptide (HSSVS), a lot of hits came out. So I'm doubting that the antibody is specific. It will be great if you could let us know how to get the immunising phosphopeptide. Thanks a lot!

ANSWER:

Thank you for your enquiry regarding ab74012 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.

The immunogen used to raise this antibody was a synthetic phosphopeptide derived from human CXCR4 around the phosphorylation site of serine 339 (H-S-SP-V-S). Unfortunately, this peptide is not commercially available.

Could you provide some further details of the protocol used and complete the following form (attached as a word document). It would be much appreciated if you could attach an image to the response.

I am particularly interested in the followings:

- sample preparation,

- blocking agent and time,

- positive control,

- secondary (no primary) control etc

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you soon and resolving this issue as soon as possible.