Anti-ABCA1 antibody [AB.H10] (ab18180)

I am still trying to do the WB for ABCA1 with the ab from abcam which the molecular weight predicted is 294Kda and I still can find the protein at that point. I send you my lab protocol ( I used 7,5% and 10% gel) and I also looked to staking and i still have no find the protein in MDA MB 231 cells. Instead i found a consistent mark at 70Kda. Could it be part of the protein????

ANSWER:

Thank you for your recent participation in Abcam's phone survey. You had mentioned that you have had a problem with one of our products (ab18100, I believe?).

I wanted to follow up with you in this regard and see if I can help to resolve the issue satisfactorily for you.
While we did provide suggestions to help improve the issues which you had experienced, we did not hear back from you as to whether these had helped.

Our products are covered by our Abpromise guarantee. If we cannot remedy your issue we will replace or refund it under our Abpromise guarantee, as you are using it according to specifications listed on our datasheet.

I look forward to hear back from you and assist you in resolving this case.

I am still trying to do theWB for ABCA1 with the ab from abcam which themolecular weight predicted is 294Kda and I still can find the protein at that point.
I send you my lab protocol (I used 7,5% and 10% gel) and Ialso looked to staking and i stillhave no find the protein in MDA MB 231 cells.
Instead i found a consistent mark at 70Kda.Could it bepart of the protein????

ANSWER:

Thank you for your reply.

I'm sorry to learn that you are still having trouble with this antibody.
It may be that the antibody is detecting a degradation fragment of the protein, and there may be some steps you can take to try to optimize the protocol.
If you want to switch to PVDF, that may be better than nitrocellulose as it has a higher binding affinity.

You may want to try a gradient gel of 4 - 20% since this protein is so large.

Since this is a multi-pass membrane protein, heating the samples at 70°C for 5-10 minutes is also acceptable and may be preferable. These types of proteins tend to aggregate when boiled and the aggregates may not enter the gel efficiently.


Large proteins will tend to precipitate in the gel, hindering transfer. Adding SDS to a final concentration of 0.1% in the transfer buffer will discourage this. Methanol tends to remove SDS from proteins, so reducing the methanol percentage to 10% or less will also guard against precipitation. Plus, lowering methanol in the transfer buffer also promotes swelling of the gel, allowing large proteins to transfer more easily. Methanol is only necessary if using nitrocellulose. If using PVDF, methanol can be removed from the
transfer buffer altogether, and is only needed to activate the PVDF before assembling the gel/membrane sandwich. You should also choose wet transfer overnight at 4oC instead of semi-dry transfer.

I hope this information helps. Please contact us with any other questions.

DESCRIPTION OF THE PROBLEM No band
SAMPLE - Species: mice - What’s cell line or tissue:Raw 264.7 and FL83B - Cell extract or Nuclear extract: cell extract - Purified protein or Recombinant protein: purified protein
PRIMARY ANTIBODY Primary Antibody - Species:mouse - Reacts against: mouse - At what dilution(s) have you tested this antibody:1:500 - What dilution buffer was used: In 5% non-fat milk in TBST (0.1%Tween20) - Incubation time: over night - Incubation temperature: 4℃ - What washing steps were done: 10min 3times in TBST (0.1%Tween20)
DETECTION METHOD ECl+
POSITIVE AND NEGATIVE CONTROLS USED No, but many researches found that Raw264.7 will express ABCA1
ANTIBODY STORAGE CONDITIONS -20℃
SAMPLE PREPARATION - What lysis buffer was used: Lysis buffer contains 0.5% Nonidet P-40, 150mm NaCl,1mM EDTA, 1mM EGTA(pH8.0), 10mM Tris(pH7.4), 1%Triton X-100 - What protease inhibitors were used: Calbiochem protease inhibitor cocktail set I .Cat#539131 - What loading buffer was used: 4X Tris-Cl/SDS (pH6.8) 7mL,Glycerol 3.8mL, SDS 1g, DTT 0.93g, Bromophenol Blue 1.2mg. - Phosphatase inhibitors :No - Did you heat the samples: temperature and time: 100℃ 10min
AMOUNT OF PROTEIN LOADED 50g and 200 g
ELECTROPHORESIS/GEL CONDITIONS - Reducing or non reducing gel: reducing - Reducing agent: DTT - Gel percentage : 5% and 7.5%
TRANSFER AND BLOCKING CONDITIONS - Transfer conditions: (Type of membrane, Protein transfer verified): PVDF membrane transfer 300mA 2hour 5. Blocking conditions - Buffer: Tris-buffered saline with 0.1% Tween 20. - Blocking agent: milk, BSA, serum, what percentage:5%milk - Incubation time:1 hour - Incubation temperature: room temperature SECONDARY ANTIBODY - Species:goat - Reacts against: mouse - At what dilution(s) have you tested this antibody: 1:2500 - Incubation time: 1 hour - Wash steps: 10min 3times in TBST (0.1%Tween20) - Fluorochrome or enzyme conjugate: HRP conjugate - 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

ANSWER:

Thank you for completing the western blot questionnaire and for including the attached western blot image.

I agree that this vial of ab18180 is not working as expected. If you have received this product within the last 6 months or so I would be happy to offer a replacement or credit.

Hi, I am using ABCA1 antibody ( cat  number 18180) and it works very well on imunnofluorescence. However, I am not being able to see the ABCA1 protein in western blot.   Have you have any suggestion in terms of Gel type, protein extraction, % of acrilnamide or blocking? I am really interested to have this results very fast, soIi am wainting for your answner.

ANSWER:

Thank you for contacting Abcam.
I am sorry to hear of the difficulties that you have been experiencing using these products. I do n ot see a specific protocol for use of this product in WB and so recommend using standard protocols. However if you would send me the details of your protocol I will look those over and see if I might be able to make suggestions to improve your results.

Please including the tissue type you are working with, your sample preparations including denaturing and reducing steps, the amount of total protein per well, the positive controls you have used, loading controls or poncheau staining results, blocking procedures, antibody incubation steps, washes, images and any steps that you may have changes while troubleshooting.
These details will not only help me to better understand your difficulties and be able to offer suggestions but will also be used for internal testing of the products if deemed necessary. If we are unable to solve the problems you are experiencing we will replace or refund the antibody according to our Abpromise guarantee.
Please let me know if you have any questions.

Feedback in the survey

ANSWER:

Thank you for providing feedback in our survey.

I am very sorry to hear that the protocol tips did not resolve the problem. As mentionned in the last email, I will therefore replace or refund the antibody for you. I apologize for this inconvenience.

I would appreciate, if you could quickly summarize the results of the last test as well- was there still the band at 440kD?

I am looking forward to hear back from you on how you would like to proceed. Thank you for your collaboration.