Anti-IGFBP5 antibody (ab4255)

Product code: 4255
Lot number: GR41492-2
Inquiry: Hi, I am using the antibody for western blot of human hela cell cytoplasmic extracts. I get two major bands, one at 37kD, and one at 50kD. I also get a very faint band at ˜30kD. The predicted MW of the protein is 31kD (as stated in the datasheet). I'm debating whether the 37kD band is specific - do you have any such experience? A few points: I used both 10% and 12% gels, blocked with milk. Thanks

ANSWER:

Thank you for contacting us and reporting the problems you have encountered in using anti-IGFBP5 antibody (ab4255). We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.
In order to understand the problems encountered more fully, I am attaching our questionnaire so that I can gather further information regarding the samples tested and the protocol used. Once I have receive the completed questionnaire, I will look at the protocol and see if there are any suggestions we can make that may improve the results. If you could attach an image of the blot obtained this would help greatly in understanding what the results have been.
This information will also allow us to investigate this case internally and initiate additional testing where necessary. I look forward to receiving your reply.

Our customer wonders whether ab4255 has homology with mouse. If you answer is positive, the customer wishes to testab4255 in WB with mouse samples. Are you interested in this type of the information for the Abreview promotion (The customer buys certain antibodies and upload Abreview on Abcam's website and then we give 100% credit note for this antibody to buy next antibody)?

ANSWER:

please find below the discount code.
Could you please send us your customer's details? email address + institute or company name, so we know when they submit their Abreview.
Thank you.
DISCOUNT CODE: ******
Expiration date: DD MM YYYY

I am very pleased to hear you would like to accept our offer and test ab4255 in Mouse (95%, see attachment). This code will give you 1 free primary antibody before the expiration date. To redeem this offer, please submit an Abreview for mouse and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. 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: www.abcam.com/collaborationdiscount.

Further to our telephone conversation this morning, please find below three scans of western blots which I have produced over the last week or two relating to your IGFBP3 and IGFBP5 antibodies. They were carried out using the ovarian cancer cell line PE04 and were denatured at 60 degrees for 60min prior to running on a 12% SDS-PAGE gel.

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The first blot above shows IGFBP5 (ab4255) and clearly shows a single band of the right mwt (it should be 31kda).

The second blot shows IGFBP3 (definately ab4248 and NOT ab10733 as I thought - I have the spec sheet) with the priamry antibody titrated down at 10microl/15mL, 5microl/15mL and 2.5microL/15mL in Roche BSA-based blocking agent.

The third blot again shows IGFBP3 (again definately ab4248 and NOT ab10733) at 2.5microl/15mL (different secondary antibody to blot 2) in (i) Roche BSA-based blocking agent (speckly one on left) and (ii) in 5% MARVEL in TBS (clear one on right). Despite blot three having a clear, single band when using 5% MARVEL, the band appears to be of approximately 60-65kda rather than the expected 41kda. The markers appear OK as the IGFPB5 in blot 1 is of the correct size.

Any comments?

Many thanks

ANSWER:

Thank you for your patience in awaiting a response. Having discussed this issue with my colleagues and the originator of the product. The following may explain the route cause of the problem

I have been told that in all cases, the IGFBP's are "sticky" and readily aggregate with themselves or other proteins even in SDS. Another somewhat unusual property that using b-mercaptoethanol is much more effective in preventing IGFPB protein interactions than DTT. I would advise the use of 5% b-mercaptoethanol in the loading buffer instead of the DTT that you have been using previously.

Please let me know how you get on with this.

ab4255-50

1. Order details: a.. Batch number: lot no: 89052 b.. Abcam order or Purchase order number: S050312 c.. Antibody storage conditions (temperature/reconstitution etc) 4?

2. Please describe the problem (high background, wrong band size, more bands, no band etc). No significant band and high background.

3. On what material are you testing the antibody in WB? Cell extract and condition medium (concentrated about 100 fold)

3. The lysate a.. How much protein was loaded: 50 ug a.. What lysis buffer was used: RIPA buffer b.. What protease inhibitors were used: leupeptin, aprotenin, PMSF, NaF, Na3VO4, EGTA c.. What loading buffer was used: 3X loading buffer d.. Did you heat the samples: temperature and time: 95?, 5min 4. Electrophoresis/Gel conditions/ Transfer conditions a.. Reducing or non reducing gel: reducing gel b.. Gel percentage : 10 % SDS-PAGE c.. Transfer conditions: 300mA, 2 hour (wet transfer) 5. Blocking conditions a.. Buffer: TBST (0.1% Tween-20) b.. Blocking agent: milk, BSA, serum, what percentage: 5 % skin milk in TBST c.. Incubation time: 1 hour d.. Incubation temperature: room temperature 6. Primary Antibody a.. Specification (in which species was it raised against): HEK293, foreskin fibroblast · At what dilution(s) have you tested this antibody: 1:1000, 1:10000

· What dilution buffer was used: 5 % skin milk in TBST · Incubation time: 2 hours for room temperature and 16 hours for 4? · Incubation temperature: 2 hours for room temperature and 16 hours for 4?

· What washing steps were done: wash 6 times and the internal is 5 to 10 minutes (wash solution including 0.1 or 0.5% tween-20 in TBS buffer)

7. Secondary Antibody a.. Specification (in which species was it raised against)? Anti-rabbit-HRP (sc- b.. At what dilution(s) have you tested this antibody: 1:3000 c.. Incubation time 2 hours for room temperature d.. Wash steps: wash 6 times and the internal is 5 to 10 minutes (wash solution including 0.1 tween-20 in TBS buffer) e.. Do you know whether the problems you are experiencing come from the secondary? 8. Detection method ECl, ECl+, other detection method: Western Lightning (NEN) 9. Background bands · Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): Yes

· Is the blocking step sufficient? Yes

· 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? No

· Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) I will e-mail the data to you. The data of image of blot in the next page.

11. Did you apply positive and negative controls along with the samples? Please specify. In our study, we had confirmed the human foreskin fibroblast cells can express the IGFBP5 mRNA by RT-PCR. But we can not determine the IGFBP5 with abcam antibody by western blotting. We test the same condition and the same samples but we used the first antibody of IGFBP5 from [competitor]. The data show we can determine the IGFBP5 in our fibroblasts.

It let us frustrate. In the data sheet of Abcam is described this antibody can apply to western blotting study but now can not work significantly in our lab. We will send some results of western blotting to you and hope you can tell us how to do. We have wasted many times to test this antibody.

10. Optimization attempts · How many times have you tried the Western? About 4-6 weeks

· Do you obtain the same results every time e.g. are background bands always in the same place? Yes · What steps have you altered? · We had tried to change the dilution fold of first antibody. We also had increased the percentage of tween-20 from 0.1% to 0.5%. And had changed the incubated condition. We had incubated the first antibody at 4? overnight.

ANSWER:

Thank you for your enquiry.

I am sorry to hear that your customer has been having difficulties with this antibody.

I have read your customers questionnaire and they have tried all the suggestions that I would recommend. They have demonstrated the expression of the RNA and protein using a competitors antibody. They have tried various washing conditions without success.

Quality is important to Abcam. I would like to offer your customer a credit note if the antibody was purchased within the past 90 days.

I look forward to hearing from you.

We had purchased the product of ab4255 and had tried in our cells. Our cell is human foreskin fibrolast and could detect the mRNA level of IGFBP5. Using this product we cannot detect the protein level on medium or cell. The medium we had culture the cell for 72 hours and collected and concentrated at least 100 fold. We don't know how to do because the backgroud of western boltting is so high. You had tried to dilute the antibody from 1:1000, 1:5000, 1:10000 but the protein of IGFBP5 still can not detect in our western menbrane but another protein we can detect such as actin and G6PD. We do not how to optimal the condition. Please help us.

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with this antiserum.

Quality is important to Abcam. According to our datasheet western blotting was tested on concentrated serum free T98G culture supernatant. In addition fragments of 23 and 18kDa are also detected.

I would greatly appreciate it if you could compete our technical questionnaire by clicking on the link below. This will better enable our technical team to combine your protocol and determine the optimisation steps that you have taken.

http://www.abcam.com/index.html?section=western&pageconfig=technical&intAbID=4255&mode=questionaire

I look forward to hearing from you.