Anti-beta Actin antibody [mAbcam 8226] - Loading Control (ab8226)

I have spoken to the customer but still another question is being passed on to me as per below:
"A replacement would be ideal, but I'd like to be sure that it will work - is there a different lot that it will be replaced with, or will it be the same one?" Please advise!

ANSWER:

I just checked the lot number your customer provided, and it looks like our system doesn't recognises it. Would you mind checking it? The number I was given is: XXXXXX. In addition, or as an alternative, I would need the PO# and the date of the order for a replacement or.

Even if I cannot check the lot number, I would like to reassure your customer that this is a very reliable antibody with only four complaints and over 150 units sold within 3 month. As the lot has past our quality control, I guess that your customer has received a faulty lot. Any replacement would be covered by our Abpromise as well.
However, we would be happy to give a credit note as well if the customer prefers this.
I hope this information is helpful to you. I am looking forward for the requested details and on how we shall proceed.

The customer has completed the questionnaire as per below (plus image attached), please have a look and kindly advise:



Thanks for that. Below is the completed questionnaire, and attached is a file with images of two example blots. For comparison, I probed for Actin on blots that had already been probed for two other antibodies that normally show similar signal intensity to Actin; as can be clearly seen, the Actin signal is extremely weak in comparison.



1) Abcam product code ab8226



2) Abcam order reference number or product batch number Lot no GR2836-5



3) Description of the problem

Very high concentration of antibody is needed to get a signal – at 1/500, several minutes exposure was necessary to see even a weak band. With previous batches of this antibody I’ve seen a strong signal at 1/2500 with exposure times of one minute or less.



4) Sample preparation:

Type of sample (whole cell lysates, fraction, recombinant protein…) Rat brain whole cell lysate

Lysis buffer RIPA

Protease inhibitors Sigma protease inhibitor cocktail P8340; AEBSF 104 mM, Aprotinin 80 uM, Bestatin 4 mM, E-64 1.4 mM, Leupeptin 2mM, Pepstatin A 1.5 mM

Phosphatase inhibitors None

Reducing agent: DTT 50 mM

Boiling for ≥5 min? yes/no No; 55C 5 min

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

Positive control n/a

Negative control n/a



5) Percentage of gel 8%

Type of membrane PVDF

Protein transfer verified Yes – successfully detected other proteins of similar size

Blocking agent and concentration 2% milk and 5% BSA both tested

Blocking time 1 hour

Blocking temperature Room temperature



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

Concentration or dilution 1/500

Diluent buffer 2% milk or 5% BSA, both in TBS with 0.05% Tween-20

Incubation time Overnight, ˜16 hours

Incubation temperature 4C



7) Secondary antibody:

Species Goat

Reacts against Mouse IgG

Concentration or dilution 1/5000

Diluent buffer 2% milk

Incubation time 1 hour

Incubation temperature Room temperature

Fluorochrome or enzyme conjugate HRP



8) Washing after primary and secondary antibodies:

Buffer TBS with 0.05% Tween-20

Number of washes 3 x 10 min



9)Detection method ECL



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

Do you obtain the same results every time? Yes

What steps have you altered to try and optimize the use of this antibody? Increasing antibody concentration and testing different blocking agents and antibody diluents

ANSWER:

Thank you for this additional information.

To be honest, your customer should have seen satisfactory results with their protocol. I would , therefore, like to offer them a replacement or a credit note.

However, I would need the original order number.

I am looking forward to your reply.

Could you kindly please have a look and advise me this particular problem I’ve received from my customer:



ab8226 (mouse monoclonal anti-beta actin)

I've used this antibody in the past and had no problems using it at concentrations down to 1/5000, but this order has worked poorly, giving weak signal even at 1/500 with a 7 minute exposure.

I'm using it in Western blot. I've used it at concentrations as low as 1/500, and tried both 2% milk and 5% BSA for blocking and antibody dilution, all giving poor results.



Please advise J

ANSWER:

Thank you for contacting us.

I am sorry to hear your customer is experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.


I look forward to receiving your reply.

Thank you for your response, I do not currently have enough volume to incubate my membrane with a 1:150 dilution, but I can use what I have left for about a 1:200 dilution, which I will incubate overnight tonight. I have a positive control of the Human Liver Microsomes in each blot, and have not had trouble detecting these bands, I am also looking into GAPDH and/or actin staining.

Also, my adviser and I have decided to go with the Anti-Cytochrome P450 2A6 antibody (ab3570), and she has already submitted the purchase order. How would we go about receiving that discount on this item?

ANSWER:

Thank you for your response.

While I cannot retroactively apply a discount to the order you have placed, the code will entitle you to xx off of one primary antibody for a future purchase. The code must be used on or before 10 October 2012.

I would like to encourage you to take advantage of our Abreview program. It takes just a few minutes to leave a review and you can collect Abpoints which you may redeem for Abcam products or Amazon gift cards while at the same time sharing information about the product with your colleagues worldwide. To leave and Abreview, go to the webpage for the product, click on the Abreview Tab right below the shipping information and clickSubmit an Abreview. You can gain more Abpoints by submitting images as well.

I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

Hello,

I am a graduate student at University of the Sciences in Philadelphia, I recently ordered and received the ab53027 Anti-CYP2A6 antibody.
I have been using it to try to detect CYP2A6 expression in both Human Liver Microsomes and microsomes I isolated from cell culture.
I have been using it at a 1:1000 dilution - 10uL in 10mL of my blocking buffer, thus far I have had no success detecting the cell's microsomes. I have been able to consistently detect the liver microsomes so I know that my blotting procedure works, the antibody is functional and that I have an appropriate secondary antibody and chemiluminescent stain.
On the product information insert it says to use the antibody at a 1/150 dilution for western blots, this seems quite high. Is it that the antibody is has weak affinity for CYP2A6 and needs to be at such a high concentration?
Also, when I attempted to visit the abcam.com website for more information I found that this particular antibody is no longer available. Is this just a supply issue, or is there a functional reason for this change?
Please advise on what volume of antibody you recommend for me to incubate with my blot, and in what volume of blocking buffer, as well as the duration of incubation i.e. 1 hour, 2 hours, overnight etc.

Thank you for your time.

Please let me know if you need any more clarification on my procedure.

Stephanie L. Schaffer
BS in Pharmacology/Toxicology 2009
MS in Pharmacology/Toxicology 2012
University of the Sciences in Philadelphia
mailto:sschaffer@mail.usip.edu
http://www.usip.edu/sga

ANSWER:

Thank you for contacting us.

The recommended dilution of 1:150 is a recommendation which we make to our customers based on our experiments or those of supplier, collaborator or customer input. Although we cannot know exactly why certain products require higher dilutions in different protocols issues of affinity or reaction conditions will play a role.

I would recommend using this product at the higher concentration recommended on the datasheet. I would also recommend running a positive control at the same time using the human liver Microsomes and strongly recommend using a loading control such as Beta Actin or GAPDH.They are used to normalize the levels of protein detected by confirming that protein loading is the same across the gel. The expression levels of the loading control should not vary between the different sample lanes. I have included links to these loading controls below.

As you have not yet seen expression in your sample cell line I would also recommend a longer incubation, 4C overnight.

We currently have a catalog of over 70,000 products. While we do make many of our products in house, we do receive others from outside suppliers. This product has been discontinued by our supplier; we do not know the exact cause for this discontinuation. We do offer a number of other CytochromeP450 2A6 antibodies which we do guarantee to work in western blot on cell lines. Our Rabbit polyclonal antibodies to Cytochrome P450 2A6, AB64890 or AB3570 may best suit your needs. As this product was discontinued shortly after your purchase I can offer you a discount off of anotherCytochrome P450 2A6 antibody should you decide to try one.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Beta Actin Loading Control:http://www.abcam.com/beta-Actin-antibody-mAbcam-8226-Loading-Control-ab8226.html

GAPDH Loading Control:http://www.abcam.com/GAPDH-antibody-mAbcam-9484-Loading-Control-ab9484.html

Further information about use of loading controls may be found at the following link:http://www.abcam.com/index.html?pageconfig=resource&rid=275

Two of our otherCytochrome P450 2A6 antibodies:

AB3570:http://www.abcam.com/Cytochrome-P450-2A6-antibody-ab3570.html

AB64890:http://www.abcam.com/Cytochrome-P450-2A6-antibody-ab64890.html