Anti-Angiopoietin 1 antibody (ab8451)

Dear
Our customer didn't test with negative control.
Please let me know what she can use as a negative control and if you can provide it to our customer.

Best regards

ANSWER:

Thank you for getting back to us. Unfortunately, xxx is away from the office today so I will deal with this enquiry.

I think what xxxx was suggesting was to perform a "no primary" control, in order to determine if the secondary antibody was making any contribution to the non-specificity observed, as well as a sample known no to express Angiopoietin 1. Unfortunately, we do not have this to provide.I would suggest your customer perform this as well as the additional suggestions Kate made:

1. I can suggest to try incubate overnight 4oC which can often provide more efficient and specific staining. I can also recommend to try a lower dilution, 1:2000 to reduce the non specific staining

2. Could you confirm the wash steps? I can suggest to wash 4 times for 5 minutes in PBS containing 0.2% Tween at the appropriate wash steps if this has not already been tried. Also, changing from PBS to TBS can provide a more stringent wash.

Additionally, could you confirm the following:

1. Could you confirm if the current vial of secondary antibody is working well with other primary antibodies?

2. Could you confirm if a loading control staining has been included to help asses the quality of the samples?

Many thanks for your continued assistance.

Product code: 8451
Lot number: GR24123
Inquiry: Multi bands The data was shown multi-bands in the same sample. I would like to know what band indicates angiopoietin-1 protein. Please advice and comment about this data.

1) Abcam product code
ab8451

2) Abcam order reference number or product batch number
GR24123

storage temperature of antibody -80oC

3) Description of the problem
Multi bands

4) Sample preparation:
Type of sample and species(whole cell lysates, fraction, recombinant protein and Human,
mouse; mouse brain

Lysis buffer RIPA buffer (Sigma)

Protease inhibitors: Protease inhibitor cocktail

Phosphatase inhibitors : no

Reducing agent: DTT

Boiling for 5 min? 10min at 90oC
Protein loaded ug/lane or cells/lane : 50 ug/lane

Positive control : no
Negative control : no

5) Percentage of gel : 4-12% Bis-Tris gels

Type of membrane : PVDF membrane
Protein transfer verified : Ponceau S

Blocking agent and concentration : 5% BSA in TBST
Blocking time : 1 hour
Blocking temperature : room temperature (RT)

6) Primary antibody (If more than one was used, describe in ¡°additional notes¡±) :
Concentration or dilution : 1:1000
Diluent buffer : 5% BSA in TBST
Incubation time : 1 hour
Incubation temperature: RT

7) Secondary antibody:
Species: goat anti-rabbit IgG-HRP (invitrogen-g-21234)
Isotype: IgG
Reacts against: rabbit
Concentration or dilution : 1:3000
Diluent buffer : 5% BSA
Incubation time : 30 min
Incubation temperature: RT
Fluorochrome or enzyme conjugate: HRP

8) Washing after primary and secondary antibodies:
Buffer : TBST
Number of washes : 3 times

9)Detection method ECL

10) How many times have you run this staining? : 4 times
-Do you obtain the same results every time? : yes
-Have you run a No Primary control? (yes/no) : no

-What steps have you altered to try and optimize the use of this antibody?

Document attachment: Attaching images of your blot is strongly recommended and can greatly speed up our investigation of your problem.

ANSWER:

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

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.

I would like to reassure you that ab8451 is tested and covered by our 6 month guarantee for use inWB and human and mouse samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

Regarding the results, there should be a single band at 57 kDa (sometimes there is a higher band at around 75 kDa which indicates glycosylated protein, but your results do not show this). Reviewing this case, I would like to offer some suggestions to help optimize the results. I would also appreciate if you can confirm some further details to assist in my investigation of this case.

1. I can suggest it would be beneficial to consider including an endogenous negative control samples. Could you confirm if this has been tried?

2. Could you confirm if the current vial of secondary antibody is working well with other primary antibodies?

3. I can suggest to try incubate overnight 4oC which can often provide more efficient and specific staining. I can also recommend to try a lower dilution, 1:2000 to reducethe non specific staining

4. Could you confirm the wash steps? I can suggest to wash 4 times for 5 minutes in PBS containing 0.2% Tween at the appropriate wash steps if this has not already been tried. Also, changing from PBS to TBS can provide a more stringent wash.

5. Could you confirm if a loading control staininghas been includedto help asses the quality of the samples?

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 details.

1. The left 6 lanes represent 6 Normal pigs. They were healthy and untreated with drugs. Themiddle6 lanes represent 6 pigs with Renal artery stenosis (RAS), but untreated with drugs. 6 lanes on the right represent RAS pigs treated with drugs. The drug was thought to preserve the kidney function. I do not know the expression level of angio-1 in normal pigs. We want to see if there is any change in these animals.Basically, there is no negative control. 2. I did not test the residual signal frompreviousblot. Theangiopoietin-1 (both ab8451 and ab95230) was tested after b-actin but before the others. That strong band around 44 kDa might represent residual signal from b-actin in picture of ab95230. But the other bands were not likely from residual signal.

ANSWER:

Thanks again for the additional data. I think there are a few confounding factors here that make the western blot data difficult to interpret confidentally: 1. I agree with you that the intense 40kDa band is likelyb-actin in both blots, but we can not be certain. 2. There also may be bleed-through from the first anti-angiopoietin blot to the second angiopoietin blot. 3. Pig angtiopoietin-1 has technically not been tested previously with either ab8451 or ab95230, so the specificity of these antibodies with respect to pig samples is not known. 4. The expression pattern ofangtiopoietin-1 in pig kidney tissue, including any alternate forms or glycosylated forms, are not known, at least based on my brief literature search. You may consider treating your samples with or without PNGase-F, an endoglycosylase which would remove complex oligosaccharides from N-linked glycoproteins. If treated samples show a band with a smaller molecular weight, this suggests the detected protein was gylcosylated and likely angtiopoietin-1. I look forward to hearing if these suggestions prove useful.

To answer your questions: 1. I have tested TGF-b, b-actin and AIF on the same membrane. They all works fine. But no other anti-angiopoietin-1 antibody was used, except Ab8451 and Ab95230. Attachment is the picture of blot with b-actin. 2. 50ug/lane protein was loaded to gel. 3. Yes, Laemmli Sample buffer was from Bio-rad (161-0737), prepared 950ul of sample buffer with 50ul b-ME. Then samples were boiled at 95 C for 5 min. 4. Yes, they are the same, cause I tested them on the same membrane, one after another with stripping, washing and re-blocking between two probes. Hope that will be helpful. Thank you very much!

ANSWER:

Thanks for the additional information. A couple more questions... 1. Are one or more of the samples from the blot considered a negative control, in that it is known to not express Angiopoietin 1? 2. After stripping but before reprobing, was it determined that there was no residual ECL signal remaining from the previous blot? Thanks again!

We are testing whole tissue homogenate from pig kidney, using proteanase inhibitor and phosphatase inhibitor. The sample was also sonicated. We used anti-angio-1 (ab8451) antibody first, and the results showed in the second picture. Then we switched toanti-angio-1 (ab95230)antibody, the results showed in the first picture. 5% dry milk in TBS w/ 0.1% Tween-20 was used to block and dilute the antibodies. The primary antibodies were diluted 1: 1000 and incubated over night at 4 degrees. The secondary antibodies were diluted 1: 10000 and incubated at room temperature for 1 hour.

ANSWER:

Thanks for providing the western blot images as well as the protocol and sample preparation details. I just have a few additional questions: 1. Was any other antibody usedsuccessfully in western blotting with these same samples? 2. How much protein was loaded per lane? 3. Angiopoietin may be glycosylated and may contain disulfide bonds. This may account for bands at higher molecular weights than expected. Was reducing agent used in sample prep? 4. Are the samples in each lane of the both blots the same, ie. is the lane 1 sample in the ab8451 blot the same sample as that run in lane 1 on the ab95230 blot? Thanks in advance for the additional information.