Anti-beta Actin antibody [mAbcam 8224] - Loading Control (ab8224)

Needs a loading control antibodyto use with C.albicans.

ANSWER:

Thank you for your call today and for your questions.

Since ab9485 and ab8224have not been tested inC.albicans samples,I can offer a discount off a future purchase if you buyone of these antibodiesnow, test it inC.albicans, and submit feedback to us in the form of an Abreview. The discount would be worth 1 free primary antibody.

If you are interested in this offer, please follow these steps:

1. Reply to this e-mail to let me know that you would like to proceed and testone of these antibodiesin C.albicans. I will then send a discount code. This code must be issued before purchasingtheantibodyso please wait for my reply before ordering.

2. Purchaseone of the antibodieseither by phone, fax, or online (www.abcam.com).

3. Test it in C.albicans.

4. Let us know the results, positive or negative, using our Abreview system (this will take about 10 minutes and images are great if you have them!). To find out how to submit an Abreview, please visit: http://www.abcam.com/abreviews.

5. After the review is submitted to us, the discount code becomes active. Simply place your new order by phone, fax, or on the web and mention the discount code. The discount can be redeemed for any primary antibody ordered and the discount code is valid for 4 months after issue.

We are always pleased to obtain feedback about our products and any information is greatly appreciated! Even ifthe antibodyturns out to be unsuitable for C.albicans, you will still receive the discount on your next purchase after your Abreview has been submitted.

Please let me know if you have any questions about this offer and I would be happy to help you further.

The Terms and Conditions of this offer can be found at: www.abcam.com/collaborationdiscount.

With the attachement.
My apologies.

We tried blocking and incubating the antibodies in 5% BSA as suggested and there are still multiple unspecific bands. The antibody is even detecting the molecular weight standard (as seen in the attached westerns, first lane). There was an improvement in in sensitivity since the exposure time was shorter due to a stronger signal. Would it be possible to try another vial of the antibody?

ANSWER:

Thank you for your reply.


The signal does appear to be a bit cleaner with the 5% BSA blocking, but there are still quite a few bands.


I would be happy to send you a free of charge replacement vial, however we only have the same lot in stock as the one you tried. Is that okay? If not, we can refund your original purchase instead or send you a different beta actin antibody, just let me know the catalogue number of the one you'd like.


I look forward to your reply.

Below you will find the completed questionnaire. If you need any other addition information please do not hesitate to contact me.


1) Abcam product code ab8224

2) Abcam order reference number or product batch number-

3) Description of the problem: Multiple bands in WB

4) Sample preparation:

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

Lysis buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 25 mM iodoacetoamide, Protease inhibitor

Protease inhibitors: 1X Protease Inhibitor Cocktail set I (Calbiochem)

Phosphatase inhibitors

Reducing agent

Boiling for ≥5 min? yes/no Yes

Protein loaded ug/lane or cells/lane 10-30 ug/lane

Positive control

Negative control


5) Percentage of gel 10%

Type of membrane: PVDF

Protein transfer verified: by pre-stained molecular weight standards

Blocking agent and concentration: 5% non-fat dry milk

Blocking time: 1h

Blocking temperature: Room temperature



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

Concentration or dilution: 1:600 - 1:2000

Diluent buffer TBS-T plus5% non-fat dry milk

Incubation time: Overnight (14h)

Incubation temperature: 4 C



7) Secondary antibody: anti-mouse (GE - from sheep)

Species: sheep

Reacts against: mouse

Concentration or dilution: 1:2000

Diluent buffer:TBS-T plus5% non-fat dry milk

Incubation time: 1h

Incubation temperature: Room temperature

Fluorochrome or enzyme conjugate: HRP



8) Washing after primary and secondary antibodies:

Buffer: TBS-T

Number of washes: 3



9)Detection method: ECL Plus (GE)

10) How many times have you run this staining? 4

Do you obtain the same results every time? yes

What steps have you altered to try and optimize the use of this antibody? Protein loading concentration (10-30 ug), antibody dilution (1:600 - 1:3000), wash condition (TBS-T +/-5% non-fat dry milk)

ANSWER:

Thank you for your reply and for confirming the details of your experiment.


I see that you're using whole cell lysates, but from what species?


Would you be able to try blocking with 5% BSA instead of milk? We do not recommend milk with this antibody as we have heard that it can lead to increased background. Block with 5% BSA for 1 hr at RT and include this block in with the primary and secondary incubations, as well.


Keep the primary at 1:2000 and incubate that overnight at 4C, but it may be best to dilute the secondary more to 1:5000. Have you run a no-primary control to see if the secondary is binding non-specifically?


Are your washes with TBST 5 mins long?


I hope the protocol suggestions improve the results that you're seeing with ab8224. I do really think that changing the blocking buffer to 5% BSA will improve the signal since we know this antibody cross-reacts with milk for some reason. If this does not improve the banding, and you are testing a guaranteed species, we would be happy to replace or refund the product for you. We look forward to your reply.

Dear sir/madam



I just received my last order, the mouse mAb against beta actin (ab8224). I think there's something wrong with the product I received, since the antibody containing solution is not very clear, and I can see small white particles in that. I asked a college of mine to check it out, and she confirmed my observation. I've not used it yet, so can not comment on it's functionality. Please let me know how you could help me.

ANSWER:

Thank you for your enquiry regarding ab8224.
This antibody is not affinity purified but IgG fractions so the storage buffer may not be completely clear. I would suggest resuspending the pellet and carry on with the experiments. In case you are dissatisfied with the results, please do not hesitate to get back to me as soon as you can.
Good luck and have a nice day!

dear Abcam,

I am searching for a house keeping antibody for Candida albicans. i have already tried 2 of them: PDI and CDC-42 from another company which were not sucessfull. What you recommend? I need an answer as soon as possible. Thanks a lot

ANSWER:

Thank you for your enquiry and your interest in our product.

We do not test our antibodies in Candida albicans on a regular basis, so it is not known if any of them would work in this species. However, the most popular loading controls in mammalian experimental system are beta actin, GAPDH, alpha tubulin.

ab8224:Anti-beta Actin antibody [mAbcam 8224] - Loading Control

- tested in:Mouse, Rat, Rabbit, Chicken, Cow, Cat, Dog, Human, Pig, Saccharomyces cerevisiae, Xenopus laevis, Fruit fly (Drosophila melanogaster), Schizosaccharomyces pombe, Chinese Hamster, Other

ab9385:Anti-GAPDH antibody (HRP) - Loading Control

- tested in: Mouse, Rat, Human, Saccharomyces cerevisiae, Xenopus laevis



If you need any further assistance in the future, please do not hesitate to contact me.