Anti-CD68 antibody [ED1] (ab31630)

it was for sure the order issued on February the 15th.
thanks!
Libero

ANSWER:

Thank you for kindly confirming these details.

As requested, we would be pleased to arrange this free of charge replacement. In order to do this, I am forwarding these details on to our distributors team who will be happy to organize this for you. This will be sent via Prodotti Gianni andI have copied them into this email so they are aware of this case.

Original order number: ######

Original purchase 1 X vial ab31630
Please provide an alternative FOCR vial ofab125212 for the customer

Thank you for your help and cooperation with this case. Please do not hesitate to contact us if you need anything further.

Tthank you very much for the info & assistance; amongst the three possibilities that you suggested, both ab76308 and ab125212 look like they'd be good choices. The latter would be more versatile, not only wb but also IHC-Fr, but I am puzzled by the specification of the minimum amount per lane in the datasheet, which is a piece of information that I am not used to see. Does it mean that ab125212 is somewhat less sentitive thanab76308? If so, I'd definitely go for ab76308.

Once we decide for one of the two, how do we proceed to arrange the replacement?

Best,

ANSWER:

Thank you for your reply.

The information regarding western blot on the datasheet for ab125212 is provided as the detection limit in western blot for this particular antibody is known to be 0.1ng/lane. For other antibodies, the detection limit is not always assessed and so this information would not be on the datasheet.

Therefore, we do not know the detection limit of ab76308 and this is not on the datasheet and so we cannot compare this to ab125212 in this way and cannot say which is the most sensitive.

Although we produce many in-house antibodies, we obtain other antibodies from a wide range of sources. Therefore, comparison experiments will not often have been done. However, I can reassure you that Abcam antibodies are all guaranteed in the applications and species listed on the datasheet.

To proceed with the free of charge replacement, please let me know which antibody you would like to receive and I can arrange it all for you via PodottiGianni.

I look forward to hearing from you. Should you have any further questions please do not hesitate to ask.

Hello Kate,
we did perform the last test, and I am afraid that the results did not change from what we had already seen.
Here are the details of the experiment:
30µg of total protein per sample were heat-denatured in the presence of loading buffer and reducing reagent, in a 4-12% NuPage gel.
The resulting nitrocellulose filter contained two lanes loaded with whole muscle lysate, prepared from murine muscle pre-damaged with bupivacaine and harvested three days post damage (it is well-established that at this stage most of the inflammatory cell population is comprised of M1 macrophages, which are CD68 positive); two lanes loaded with total cell lysate prepared from two murine macrophage cell lines (raw and J774), one lane loaded with total cell lysate from murine myoblasts C2C12.
We performed an overnight saturation with TBS-T milk5% at 4 degrees, followed by one hour incubation with primary ab (1:250 in saturating solution), 3 washes in TBST, one hour incubation with secondary ab (1:2000 in saturating solution).
As you can see from the image, in the 37kD range (where the band should be) there is no clear signal present in the muscles or in the cultured macrophages and absent in the C2C12.

At this point we can safely assume that this particular ab does not work for us; we absolutely need to quantify the M1 macrophages in our samples and I was wondering if you could suggest any other alternative from your catalog, which could then be sent to us as a replacement instead of issuing a refund.

thank you in advance for your assistance,

ANSWER:

Thank you for your reply and for kindly confirming these details.

I would like to reassure you that we monitor feedback closely on a weekly basis and we are not currently concerned about the general quality of this antibody or this batch. Regrettably, I can suggest you have received a bad vial on this occasion.

Therefore, we would be pleased to provide a replacement from the same antibody, which will still be covered by our guarantee. Alternatively, I fully understand your concerns and if you prefer an alternative replacement or credit note in this case, I will be pleased to arrange this for you.

For alternatives, we have the following CD68 antibodies tested in mouse and western blot (please note these are not mouse monoclonal antibodies):

ab53444 Anti-CD68 antibody [FA-11]
Rat monoclonal
Flow Cyt, IHC-Fr, IHC-P, IP, WB
Reacts with: Mouse
http://www.abcam.com/index.html?datasheet=53444 (or use the following: http://www.abcam.com/index.html?datasheet=53444).

ab125212 Anti-CD68 antibody
Rabbit polyclonal
IHC-Fr, IHC-P, WB
Reacts with: Mouse, Rat
http://www.abcam.com/index.html?datasheet=125212 (or use the following: http://www.abcam.com/index.html?datasheet=125212).

ab76308 Anti-CD68 antibody [EPR1392Y]
Rabbit monoclonal
WB
Reacts with: Human, Mouse, Rat
http://www.abcam.com/index.html?datasheet=76308 (or use the following: http://www.abcam.com/index.html?datasheet=76308).

I hope this will be helpful. I look forward to hearing from you with details of how you would like to proceed.

Hello again,

I am aware that we do not have a perfect blot as far as background bands are concerned, but you will agree with me that the chance of the specific band to migrate *exactly* together with one of the non-specific bands is not very high.
At any rate, we are going to try once more to run a gel in denaturing conditions; should we once again fail to detect any bands in the expected area of the blot then I will conclude that the problem is linked to the primary ab and I will expect AbCam to fulfill its warranty.
I will let you know as soon as we have the results,
talk to you soon

ANSWER:

Thank you for your message and for your cooperation.

I appreciate the time you have spent on these experiments in the laboratory.I fully understand your concerns and it is disappointing the results have not been successful. I would like to reassure you that If the results are not improved using full denaturing and reducing conditions, I will be pleased to provide a free of charge replacement or credit note as requested.

We do often find that suggesting some scientifically thought out optimization tips helps to improve the results. So I hope you can understand that we like to offer the best technical support possible to provide a satisfactory outcome.

I look forward to hearing from you with the results.

Order details:
Antibody code:
ab31630

Problem
Non-specific band Multiple bands No signal or weak signal High background

Lot number GR57516-5

Purchase order number (The Italiandistributor will have the order number reference)
or preferably Abcam order number:
Prodotti Gianni n.23


General Information
Antibody storage conditions (temperature/reconstitution etc)
4oC


Description of the problem (high background, wrong band size, more bands, no band etc.)
The antibody gives many specific bands, but the desired weight is never detected.


Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)
Regenerating mouse muscle, whole protein extract from muscle sections

Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)
Buffer: 62.5 mM Tris-HCl [pH 6.8], 10% glycerol, 2.3% sodium dodecyl sulfate [SDS], and Roche protease inhibitors.


Amount of protein loaded
25 ug


Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)
Non reducing, invitrogen precast gel 4-12%


Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)
Transferred at 30 V cost 2 hours with 48 mM Tris, 39 mM glycine, 0.04% SDS, 10% methanol. Blocked in 10& milk in TBST


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Anti CD-68 Abcam antibody code ab31630, diluted 1:250 in TBST 5% milk, incubated 1 hour at RT.
3 washes 10 minutes each in TBST.

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Santa Cruz goat anti-mouse IgG-HRP, 1:3000 in TBST, incubated 1 hour at RT.
3 washes 10 minutes each in TBST.

Detection method (ECL, ECLPlus etc.)
Santa Cruz Western blotting luminol reagent

Positive and negative controls used (please specify)
Negative control: C2C12 cells protein extract
Positive control: regenerating mouse muscle protein extract


Optimization attempts (problem solving)
How many times have you tried the Western?
4 times


Have you run a "No Primary" control?
Yes

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


What steps have you altered?
We tried to change the buffer (PBST), we tried to saturate with BSA, we tried to saturate with another anti-mouse antibody.

ANSWER:

Thank you for taking the time to complete our questionnaire and contact us.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

Reviewing this case, I would like to offer some suggestions to help optimize the results from ab31630. I would also appreciate if you can confirm some further details:

1. I am sorry we are not able to trace the order number provided in our records. I can recommend to pleasecheck with Podotti Gianni. They will be able to confirm the order number reference they were provided by us.

We do have an order for this antibody fromPodotti Gianni dated 15th Feb. Is this likely to be the correct order?

2. I can recommend to aliquot and freeze the antibody as directed on the datasheet. This will ensure it remains stable and that we can continue to provide our Abpromise guarantee.

3. Please provide more information on what samples are in each well of the images. I would appreciate if you are able to send this again, with the molecular weight markers annotated. Are you able to provide the results of the no primary control to compare this too?

4. I can recommend to try RIPA lysis buffer, rather than Tris HCl buffer. This can often provide a better protein preparation.

5. Try reducing and denaturing conditions. This will ensure the protein is in the correct conformation to run at the correct molecular weight.

6. If the secondary antibody alone is giving non specific staining, then reducing the concentration of secondary used may help to improve the results. I would suggest to try this before changing the blocking, as the blocking procedure should be working.

I can recommend that you will need to optimize the secondary antibody conditions until you obtain a completely negative result from the no primary control. If this does not provide a clear signal, you may like to consider contacting the supplier.

7. Adding 5% BSA or milk to the antibody dilution buffers can sometimes help to reduce background.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested informationand details of how you would like to proceed.