Rabbit anti-Sheep IgG H&L (AP) secondary antibody (ab6748)

I dont know if this should be posted via your webpage or not? Nevertheless, here is our answers to your questions:

We have not tested ab6748 without any blocking agent and you are right this would also be a nice control to do. On the other hand we have obtained negative results with 4 different blocking agents and therefore we seriously believe that there is something wrong with this antibody. To our experience in is normal to have problems with maybe 1 or 2 blocking agents but not with 4 different.

No permeabilising agent was used.

As mentioned previously the delution buffer was TBS (Trizma Base-sodiumchloride pH 7,4. Trizma base 6,06g and Sodium chloride 8,76g / l)

We would appreciate to get this antibody (ab6748) to work together with a primary antibody also bought from Abcam (Anti-acetyl lysine ab76). We would therefore suggest that you supply us with the protocol originally used to test the ab6748 for IHC together with another batch of the ab6748 free of charge. We would then perform an experiment comparing the two batches of ab6748 and supply you with the result. This should be in your favour as well as there could be other customers having problems with the 67077 batch.

ANSWER:

Thank you for your enquiry.

My colleague Dr Sarah Mardle is out of office until Thursday morning. As soon as she gets back, she will get in touch with you and deal with your enquiry.

Thank you for your patience.

BATCH NUMBER 67077 ORDER NUMBER 71216

DESCRIPTION OF THE PROBLEM We have recently used your antibody ab6748 (Rabbit polyclonal to Sheep IgG (alkaline phosphatase)) and have serious problems with it.

Initially we used it as a secondary antibody against another product of yours Anti-acetyl Lysine ab76 (Sheep polyclonal to acetyl Lysine). We obtained intensive staining on all the used human testis samples including controls omitting primary antibody. This indicated problems with the secondary antibody. Therefore we made a second experiment designed as follows:

Only your secondary antibody ab6748 was used in 1:1000 or 1:500 diluted in TBS. We used 4 different blockades: Human Serum (1:4), 2% normal goat serum, BSA 1:5, and Mouse serum 1:10 before applying your antibody. Sections of testicular seminoma fixed in PFA or formalin were used

In short: Deparaffination. Microwave treatment with Citrate buffer. Water, TBS. Blockade with the above mentioned. TBS 3x2min. Incubation with antibody ab6748 in TBS, 30 min RT. TBS 2x5min. Relevation buffer (0.5 M TRIS pH 9.5, 0.5 M NaCl, 0.2 M MgCl2) 5min. Development in dark with BCIP /NBT (Levamisol) 10min. This protocol is routinely used twice a week in the lab and have in the last 5 years generated good results.

The result was as follows (both fixations reacted equally).

antibody ab6748 1:1000 or 1:5000, Human Serum: Serious background staining.

antibody ab6748 1:1000 or 1:5000, 2% goat serum: Serious background staining and background respectively. antibody ab6748 1:1000, BSA: background. antibody ab6748 1:5000, BSA: slight background and background at the edge of sections.

antibody ab6748 1:1000 Mouse serum: background. antibody ab6748 1:5000 Negative to sleight background in the edge of sections.

Controls: Omission of antibody ab6748 and all combinations of blockades (Human Serum (1:4), 2% normal goat serum, BSA 1:5, and Mouse serum 1:10) were all negative.

As a result of these results (and the initial experiment) we find it problematic to continue our experiments as something seems to be wrong with this antibody. Furthermore in your application sheet you recommend dilutions in the range of 1:100 to 1:1000 for IHC, although blockade with mouse serum and antibody ab6748 1:5000 gives a slight possibility for successful experiments, we regard this inadequate for a commercial antibody and tend to believe that something is wrong with this antibody: antibody ab6748 lot 67077

Yours sincerely

SAMPLE Human testicular samples

PRIMARY ANTIBODY Anti-acetyl Lysine ab76 (Sheep polyclonal to acetyl Lysine)

SECONDARY ANTIBODY ab6748 (Rabbit polyclonal to Sheep IgG (alkaline phosphatase))

DETECTION METHOD IHC

POSITIVE AND NEGATIVE CONTROLS USED omitting primary antibody

SAMPLE PREPARATION Deparaffination. Microwave treatment with Citrate buffer. Water, TBS. Blockade with the above mentioned. TBS 3x2min. Incubation with antibody ab6748 in TBS, 30 min RT. TBS 2x5min. Relevation buffer (0.5 M TRIS pH 9.5, 0.5 M NaCl, 0.2 M MgCl2) 5min. Development in dark with BCIP /NBT (Levamisol) 10min.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 12 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes

WHAT STEPS HAVE YOU ALTERED? Taken away primary Ab and used different blockades and dillutions

ANSWER:

I'm very sorry to hear you are having problems with ab6748. We take your complaint very seriously and would appreciate if you could please clarify whether you have tested the antibody with no blocking at all and tell me what permeabilising agent has been used as well as the dilution buffer used?

My apologies for the delay, I look forward to hearing from you,