Anti-Tubulin antibody [YOL1/34] - Microtubule Marker (ab6161)

WB with yeast (S. cerevisiae)
no band
Ab: 1/1000 o/n 4 degree
10-20 ug protein loaded
detects other proteins
blocking: 5% milk
tried 2 different 2nd Abs (same results: no band)

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number xxx.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

What plant species was this antibody tested in? Do you have the accession number for the sequence the immunogen peptide is based on? I am looking to use this antibody in tomato.

ANSWER:

Thank you for your enquiry.

I received the following reply from the originator of the antibody: This antibody was raised against yeast tubulin. It has been shown to work with tubulin from all sources including plant and animal.

Therefore, this antibody should cross-react with tomato. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM no bands

SAMPLE Saccharomyces cerevisiae, whole cell extract

PRIMARY ANTIBODY Abcam rat monoclonal anti-tubulin diluted in TBS + 0.1%tween (TBST), 1/1000 dilution 2 hour incubation, 5x 5min washes with TBST

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED none, this is a loading control

ANTIBODY STORAGE CONDITIONS -20C

SAMPLE PREPARATION Lysis buffer (125mM NaCl, 25mM Tris, 2.5mM EDTA, 1%Triton-X) Protease inhibitors- aprotinin, leupeptin, pepstatin Heat samples at 95C in sample buffer for 5 minutes

AMOUNT OF PROTEIN LOADED 20 ug

ELECTROPHORESIS/GEL CONDITIONS reducing conditions, 8% gel voltage- 100V

TRANSFER AND BLOCKING CONDITIONS 1 hour wet transfer as per instructions at 15 volts for 1 hour Buffer (30g Tris, 3g Glycine, 200ml(20%) MeOH per 1L of H20) Block with Tris Buffered Saline + .1% tween + 5% nonfat powdered milk

SECONDARY ANTIBODY 45 min. incubation with anti-rat secondary antibody 1/10000 dil(Jackson labs) 5x 5 min washes TBST

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? none

ADDITIONAL NOTES This protocol has worked very well with this antibody before when we were using a previous batch.

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with this antibody. We have received favourable feedback from our customers that have used this antibody as a loading control in S.cerevisiae. I have read your technical questionnaire and I have a few comments.

Firstly I would appreciate it if you can tell me whether you have used this antibody in conjunction with another to verify the integrity of your yeast samples. Can you also tell me whether you verified successful protein transfer using ponceau as I am surprised by the total lack of signal.

Could you also provide me with details of the batch numbers that you have been having difficulties with and the batch that you have had success with previously.

I look forward to hearing from you.

1.) The sample run on the western blot was FRTL-5 (rat thyroid cells)whole cell lysate the lysate was prepped with a 1% triton x 100 lysis buffer. approximately 13 micrograms of protein was run out on the gel and transfered to PVDF membrane 2.)We probed the western blot with ab6161(anti-tubulin) microtubule antibody 1:1000 dilution in TBST/5%nonfat dry milk for 2 hours at RT with gentle agitation. We washed 3 X 5 minutes with TBST. We then treated the blot with 1:2000 anti-rat IgG HRP conjugate secondary antibody for 1 hour at RT with gentle agitation. We repeated the washes and used Amersham ECL plus to visualize the blot. 3.) No bands and little background appeared on film even after o/n exposure to film. 4.) These samples had previously been run and blotted with anti-Bidh anti-body and we had seen bands and we needed the rat anti-tubulin to confirm protein loading concentrations. 5.) The anti-body was fresh and had just been recieved from Abcam. 6.)Can you help us? Currently we are going to reprobe the blot with a higher concentration of both the rat anti tubulin and higher concentration of secondary antibody.

ANSWER:

Thank you for your enquiry. I am sorry to hear that you have been having problems with ab6161.

Your approach using a rat antiserum against a rat lysate has been shown to be successful on the western blot image on the data sheet. On this occasion 20ug of rat brain lysate was used to good effect.

Please can you confirm that you verified the transfer of the protein using Ponceau. I was also concerned that perhaps given that tubulin is tightly complexed in the cytoskeleton, it is not extracted successfully using Triton. I was wondering whether you would consider using RIPA. It may also be useful to include a HeLa whole cell extract, extracted using RIPA. This will give a better indication of whether the antiserum is to blame.

I think that increasing the mass of protein as you have mentioned is a good idea, but I am not certain that increasing the concentration of the antiserum will make a significant difference. However, it is worth trying in conjunction with the modifications/controls that I mention above.

I hope this information helps. Please do not hesitate to contact me should you require further assistance. Good luck in your research.

1. No signal (no bands) on western blot

2. Yeast cell extract (S. cerevisiae)

3. Lysates were prepared with protease inhibitors. 3OD worth of cells were used to make the lysate and 15% of the lysate was loaded on the SDS-PAGE gel. The protein level was not quantitated but there were sufficient levels of overall protein to be seen by ponceau staining. Also, probing of the blot with another antibody gave normal levels of signal. (I am attempting to use the tubulin antibody as a loading control).

4. Primary antibody was the tubulin antibody purchased from abcam. It is a rat monoclonal. Dilutions tried have been 1:1000 and 1:2000. The blot is blocked in 5% milk in TBST (10mM Tris pH8, 0.15 M NaCL, 0.05% Tween 20) for 1 hr at room temperature before an overnight incubation at 4 degrees with the primary antibody (in 5% milk TBST). The blot is then washed in TBST 5 times of 5 minutes each before the secondary antibody, also in 5% milk is added.

5. Two different anti-Rat HRP coupled secondary antibodies were tried. These secondaries have worked with other primary antibodies. The dilution used was 1:2000. The blot is incubated with the secondary for 30-60 minutes at room temperature. The blot is then washed 5 times of 5 minutes each in TBST and washed once for 5 minutes in TBS (same as TBST but no Tween) before being developed.

6. Horseradish peroxidase is coupled to the secondary and allows visualization of the signal. I'm using the PIERCE supersignal west pico chemiluminescent substrate solution for developing the blot.

7. There are no background bands on the blot. The protein cannot have been run off as yeast alpha tubulin is 49.8 and 49.7 kD (Tub1 and Tub3). The blot probed for a protein of 55.2kD shows that it is in the middle of the blot.

8. The western has been tried four times, the first two altering the secondary antibody and the second two altering the concentration of the primary antibody used. In all cases there was no signal.

It would be great if you could response as soon as possible as we are in urgent need of a loading control for the blots. Thanks!

ANSWER:

Thank you for sending me the details of your protocol. We have had no other complaints concerning this particular lot but I can send you a replacement lot free of charge. What was the original Abcam order number? Or, what PO# was used for this order? Please let me know and then I can send you another vial.