Anti-PIWIL4 antibody (ab21869)

Thanks for your kindly reply, after I contacting with this customer and passed these suggestion to her, then she modified the experiment step as your suggestion, but it did not improve the result. I attached his data in this letter, would you please offer other suggestions to her? Thanks for your kindly help.  

ANSWER:

Thank you for your message and for providing this further information.

I am sorry to hear the suggestions made have not improved the results on this occasion. I appreciate the time you have spent on these experiments and would be pleased to arrange a replacement (with a different lot number or an alternative product), or a credit note in compensation.

I look forward to hearing from you with details of how you and your customer would like to proceed.

LOT NUMBER 841630

DESCRIPTION OF THE PROBLEM

No signal or weak signal

SAMPLE

•Species: Mouse

•What’s cell line or tissue: GC-2, mouse spermatocyte

•Cell extract or Nuclear extract: Cell extract

•Purified protein or Recombinant protein: cell lysate

PRIMARY ANTIBODY

•Species: Rabbit

•Reacts against: mouse piwil4

•At what dilution(s) have you tested this antibody: 2ul/ml

•What dilution buffer was used: TBST with 0.1% BSA or 5% milk

•Incubation time: O/N

•Incubation temperature: 4℃

•What washing steps were done: wash 5min for three times with TBST

DETECTION METHOD

ECl

POSITIVE AND NEGATIVE CONTROLS USED

Yes, I loaded the positive control, mouse testis tissue (500ug), along with the samples.

ANTIBODY STORAGE CONDITIONS

-20℃

SAMPLE PREPARATION

•What lysis buffer was used: RIPA buffer

•What protease inhibitors were used:

•What loading buffer was used: 6X SDS sample buffer

•Phosphatase inhibitors: no add

•Did you heat the samples: temperature and time: yes, 95℃ for 5 min

AMOUNT OF PROTEIN LOADED

24ug

ELECTROPHORESIS/GEL CONDITIONS

•Reducing or non reducing gel: reducing gel

•Reducing agent: DTT

•Gel percentage : 7%

TRANSFER AND BLOCKING CONDITIONS

•Transfer conditions: (Type of membrane, Protein transfer verified): PVDF membrane

•Buffer: TBST

•Blocking agent: milk, BSA, serum, what percentage: 5% milk

•Incubation time:1 hour

•Incubation temperature: RT

SECONDARY ANTIBODY

•Species: Goat

•Reacts against: Rabbit

•At what dilution(s) have you tested this antibody: 0.4ug/ml

•Incubation time: 1 hour

•Wash steps: wash 5min for 10 times with TBST

•Fluorochrome or enzyme conjugate: HRP

•Do you know whether the problems you are experiencing come from the secondary? no

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3

HAVE YOU RUN A "NO PRIMARY" CONTROL? No

DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED?

Modify the transfer time and Ab dilution

ANSWER:

Thank you for taking time 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 ab21869 :

- When testing our antibodies, our lab uses 5% BSA as a blocking reagent, so I recommend switching to this instead of milk. Some antibodies give stronger, more specific signals on blots blocked with BSA instead of milk, so doing this may improve the results you are seeing, and reduce the non-specific bands. An example of the above is the western blot obtained with the Abcam GAPDH antibody ab9385 : Click here for the western blot image using ab9385 (or use the following: www.abcam.com/ab9385).

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 would like to know how old these mice were you used the testes from.

ANSWER:

Thank you for your enquiry.

The mice for catalog number ab4027, the product used in the Western blot on the datasheet for ab21869, are adults, approximately 3 months old.

Please let me know if you have any more questions.

BATCH NUMBER 156996 ORDER NUMBER 145613

DESCRIPTION OF THE PROBLEM Non-specific band, no band where Miwi2 is expected

SAMPLE testis cell extract

PRIMARY ANTIBODY ab21869: 0.5mg/ml, diluted in western washbuffer (see above), incubation for 2h, 5x 5min washes with western washbuffer before adding secondary ab

DETECTION METHOD ECL

ANTIBODY STORAGE CONDITIONS -20 ?C

SAMPLE PREPARATION tissue was dissected, immediately snap frozen in liquid nitrogen and stored in -80?C for 2 days. lysis buffer: 50mM Tris HCl pH 7.4, 1% Triton X-100, 0.2% sodium deoxycholate, 0.2% SDS, 1mM PMSF, 0.25mM AEBSF, Roche complete prot. inhibitors according to the manufacturer's protocol. 300 ul lysis buffer / 5 mg tissue tissue was homogenized with a homogenizer, then incubated for 1h (4?C, on rotation) sample was centrifuged @ 12000 rpm for 20'

AMOUNT OF PROTEIN LOADED different amounts loaded

ELECTROPHORESIS/GEL CONDITIONS 10% denaturing SDS PAGE

TRANSFER AND BLOCKING CONDITIONS transfer semi-dry on nitrocellulose membrane, 5V, 3h, buffer: towbin blocking: 10% milkpowder in western washbuffer (30mM Tris pH 7.5, 150mM NaCl, 0.25% Tween-20)

SECONDARY ANTIBODY monoclonal anti-rabbit, peroxidase conjugate [another company] 1:5000, diluted in washbuffer (see above)for 1h, afterwards 5x 5min washes

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? lysis buffer (more prot inhibitors), incubation time of lysis buffer after homogenization, amount of protein loaded

ANSWER:

I'm sorry to hear you are having a problem with ab21869. This antibody has been tested in mouse samples and if your testis cell extract are from mice I would expect ab21869 to work well for you.

I would like to suggest the following modifications to your protocol: -change the blocking buffer, concentration and incubation time; we typically recommend to try 5%BSA in TBST for 1 hour then incubate the primary antibody in TBST or TBST/BSA (it is worth trying both) and to incubate the secondary antibody in TBST only. I think the problem is likely to be due to the high concentration of the blocking agent as milk should be used at 5%. -I would also recommend using the antibody more diluted and incubate longer, for a slow targeted binding to the protein of interest (e.g. 1-2ug/ml overnight, you may find you can dilute the antibody more). We recommend using ECL+ or even more sensitive detection kits (e.g. Pierce super signal) and can I please suggest checking that your protease inhibitors and buffers are fresh and running a no primary control?

If you still experience problems with those changes please do not hesitate to contact me again as the antibody is guaranteed by the Abpromise and I can offer you a replacement or refund,