Anti-Bmi1 antibody (ab38295)

Dear tech,

Please check below reply from the customer .Thank you!

1.) Can you confirm that the bmi1 protein is present in theMHCC97-H cellline? (Is there for example a publication describing this?)

Yes, I have confirmed that bmi1 protein is present in theMHCC97-H cellline by Immunofluorescence, just as the picture attached:

2.) I can stronly recommend to use also an other blocking buffer, as changing the blocking buffer can significantly improve the results. I can recommend to use BSA as an alternative blocking buffer. Please see image on the datasheet of ab9385 as an illustration.

I have done what you advised, but it still didn’t work.

3.) Can you please let me know whether the samples have been reduced? If not, I can recommend to do so, as the antibody has been tested under these conditions.

The samples were not reduced, for I have tried different samples. And for an antibody for another protein, the result was good.

4.) Lastly, can you please confirm that the secondary antibody used is known to work?

The secondary antibody used was commonly used in my lab, and it did work well.

Best regards,

ANSWER:

Thank you very much for following this up with the customer. I am sorry that the change in the blocking buffer did not improve the results.

I would still like to recommend to the customer to reduce the samples for the Western blot either with DTT or beta Mercaptoethanol. Indeed, not all proteins need to be reduced to be visible in the Western blot and I am not sure whether it is necessary to reduce the samples to see a nice band of BMI1. As however the BMI1 is a highly complexed protein (http://www.uniprot.org/uniprot/P35226) and we have tested it under reduced conditions, I think there is the possibility that reducing the samples could solve the problem.

As I understand howeverthe frustration the customer must be experiencing, I will provide a credit note for this antibody. Please do let me know whether the customer would like to accept this.

Thank you for your cooperation. I am looking forward to hear back from you.

1) Abcam product code ab38295

2) Abcam order reference number or product batch number


3) Storage conditions:Store at +4°C.

4) Description of the problem (high background, no band, wrong band size...)

no band

5) Sample preparation:

Name of the tissue/cell line:hepatocellular carcinoma cell MHCC97-H

Sample species:human

Type of sample (whole cell lysates, fraction, recombinant protein…):whole cell lysates

Lysis buffer RIPA

Protease inhibitors: Aprotinin Leupeptin PMSF

Phosphatase inhibitors

Reducing agent

Boiling for ≥5 min? yes/no yes

Protein loaded ug/lane or cells/lane 40ug

Positive control NO

Negative control NO



6) Percentage of gel 10%

Type of membrane PVDF

Protein transfer verified

Blocking agent and concentration 5% milk in TBST

Blocking time 1 hour

Blocking temperature room temperature



7) Primary antibody (If more than one was used, describe in “additional notes”) :

Concentration or dilution at 1:1000 and 1:500

Diluent buffer in TBST

Incubation time overnight

Incubation temperature: at 4°C



8) Secondary antibody: Goat anti-Rabbit IgG H&L (HRP) secondary antibody
Species:Goat

Reacts against: Rabbit

Concentration or dilution 1:2000

Diluent buffer in TBST



Incubation time 2 hours

Incubation temperature:room temperature



Fluorochrome or enzyme conjugate: enzyme conjugate



9) Washing after primary and secondary antibodies:

Buffer in TBST

Number of washes every time for 10 minutes and 3 times



10)Detection method ECL



11) How many times have you run this staining? 5 times

Do you obtain the same results every time? yes

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

ANSWER:

Thank you for taking time to liaise with the customer and 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 ab38295. I would also appreciate if you can confirm some further details:

1.) Can you confirm that thebmi1 protein is present in theMHCC97-H cellline? (Is there for example a publication describing this?)

2.) I can stronly recommend to use also an other blocking buffer, as changing the blocking buffer can significantly improve the results. I can recommend to use BSA as an alternative blocking buffer. Please see image on the datasheet of ab9385 as an illustration.

3.) Can you please let me know whether the samples have been reduced? If not, I can recommend to do so, as the antibody has been tested under these conditions.

4.) Lastly, can you please confirm that the secondary antibody used is known to work?


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.

I'm working on Psc and Su(z)2, which are the Drosophila homologues of Bmi-1. I would be interested in knowing whether any of your Bmi1 antibodies (ab25791, ab38295, and ab14389) cross react with either of the Drosophila homologues. Would it be possible for me to test a 1:1,000 dilution of each of the antibodies on WBs for cross- reactivity? Alternatively, I could send you a blot to test for yourself. I'm happy to buy any ab's that cross-react, but I can't justify spending $300/ab without knowing whether any of them will cross-react...

ANSWER:

Thank you for your enquiry. I performed a homology search of the immunogen of ab25791 and ab14399 and of the whole human protein with the drosophila protein and unfortunately found that it is so low that it is not detected by a BLAST search. I therefore do not think those antibodies will detect the Drosophila protein and recommend to purchase Drosophila specific antibodies.

I'm sorry we currently do not have those in our catalogue and wish you all the best in your research,