Anti-TIMP3 antibody - Loop 1 (ab39184)

Can you confirm that this material is in 0.25M Sodium chloride? Our Chemist cannot seem to see this on the COA or Data sheet. Please advise as soon as possible,

ANSWER:

Thank you for contacting Abcam. I can confirm there is 250mM sodium chloride in the storage buffer for ab39184. Please let me know if there is anything else I can help you with.

LOT NUMBER GR41994-1

ORDER NUMBER 39180

DESCRIPTION OF THE PROBLEM Multiple bands

SAMPLE lanes 1,2, 3 and 4: human lung cancer cell line (Calu6)whole cell lysate, lanes 5 and 6: normal mammary epithelial cells (MCF10A)whole cell lysate,

PRIMARY ANTIBODY anti-TIMP1 (ab61224) diluted 1/200 in PBS-Tween 0,1%, incubation ON at 4 celcius washed 4 times 5 min with PBS-Tw 0,1%

DETECTION METHOD Enhanced Luminol (Perkin Elmer) film X-OMAT exposed 2 and 30 min

POSITIVE AND NEGATIVE CONTROLS USED We consider MCF10A cells as positive control

ANTIBODY STORAGE CONDITIONS aliquot the same day it arrived at the lab and stored at -20

SAMPLE PREPARATION lanes 1,2, 3 and 4 lysing buffer: HEPES 25mM pH7,5, NaCl 150mM, MgCl2 10mM, EDTA 1mM, Trition 0,1%, Glycerol 10%, aprotinin 10microM and sodium vanadate 1mM lanes 5 and 6 lysing buffer:sodium phosphate 20mM, NaCl 150mM, Triton 1%, EDTA 5mM, PMSF 0,2mg/ml, Aprotinin 10microg/ml, leupeptin 10microg/ml, sodium vanadate 0,25mg/ml Sample buffer 2X: Tris 150mM pH 6,8, SDS 1,2%, Glycerol 30%, beta-mercaptoethanol 15%, bromophenol blue 0,0018% Protocole: samples were mixed with sample buffer 2X (1:1) and boiled for 10 min

AMOUNT OF PROTEIN LOADED lanes 1,3 and 5 70microg, lanes 2,4 and 6 20microg

ELECTROPHORESIS/GEL CONDITIONS 12% resolving gel for denaturing SDS-PAGE and 4% stacking gel

TRANSFER AND BLOCKING CONDITIONS wet transfer with Tris-Glycine-SDS buffer with 20% methanol, 900mA for 90min Blocking: PBS-Tween 0,1%- milk 5%, 90min, RT

SECONDARY ANTIBODY anti-Rabbit-HRP (Santa Cruz sc-2004) 1/10000 in PBS-Tw 0,1% 90min washed 4 times 5 min with PBS-Tw 0,1%

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? No

WHAT STEPS HAVE YOU ALTERED? We changed sample loading buffer. The one before contained less beta-mercaptoethanol and hight molecular weigth appeared on film

ADDITIONAL NOTES Is it possible that we detect dimers of TIMP1

ANSWER:

Thank you for taking time to complete our questionnaire. I am sorry to hear that antibodies ab16123, ab39184 and ab61224 are not providing satisfactory results.

The details provided will enable us to investigate these cases and will provide us with vital information for monitoring product quality.

Having reviewed these 3 cases, I would like to offer some suggestions to help optimize the results from ab16123, ab39184 and ab61224 :

To reduce the background, I recommend to load a maximum of 20µg of samples on the gel. I would suggest to use each primary antibody at a dilution 1/1000 and to dilute each one in the blocking solution. The dilution factor can then be optimized around this value. I would also recommend to try an incubation at room temperature for 2 or 3 hours. Some antibodies bind stonger and more specifically at room temperature. In order to determine the origin of the background I would recommend to run a no primary control in order to see if the background is due to some non-specific binding by the secondary antibody. Last but not least, 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 : www.abcam.com/gapdh-antibody-hrp-loading-control-ab9385.html#GAPDH-Primary-antibodies-ab9385-3.jpg . 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.

LOT NUMBER Gr 8727-1 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM I'm obtaining bands above 30 kDa for ab39184 and ab39185 (TIMP antibodies). In the description of the antibodies ab39184 and ab39185 is: Anti-TIMP3 antibody - Carboxyterminal end (ab39185) WB: 1/1000 - 1/5000. Used under non reducing conditions. Detects a band of approximately 21 kDa (predicted molecular weight: 24 kDa). Anti-TIMP3 antibody - Loop 1 (ab39184) WB: 1/1000 - 1/5000. Dilution optimised using Chromogenic detection. Detects a band of approximately 24 , 30 kDa (predicted molecular weight: 24 kDa). My bands are above 30 KDa, what should I do? SAMPLE Type of sample: cell lysates of mouse PRIMARY ANTIBODY Primary antibody (If more than one was used, describe in “additional notes”): only TIMP3 (ab 39184) Concentration or dilution: 1:200 Diluent buffer: TTBS 1x (TBS 10x and distilled water) Incubation time: overnight Incubation temperature: 8ºC DETECTION METHOD ECL ANTIBODY STORAGE CONDITIONS as recommended on the datasheet. SAMPLE PREPARATION Lysis buffer: RIPA Boiling for ≥5 min? yes AMOUNT OF PROTEIN LOADED Protein loaded ug/lane or cells/lane: 30 ug/ protein ELECTROPHORESIS/GEL CONDITIONS Non reducing gel. Percentage of gel: gradient 5-20% TRANSFER AND BLOCKING CONDITIONS Type of membrane: nitrocellulose membrane Protein transfer verified: yes Blocking agent and concentration: milk 5% Blocking time: 1 hour Blocking temperature: room temperature SECONDARY ANTIBODY Secondary antibody: anti-rabbit IgG HRP conjugated ( Amersham NA934) Species: Reacts against: rabbit Concentration or dilution: 1:5000 Diluent buffer: TTBS 1x Incubation time: 1h Incubation temperature: room temperature Fluorochrome or enzyme conjugate: HRP Washing after primary and secondary antibodies: Buffer: TTBS Number of washes: primary, 3x (5 min.) secondary, 3x (15 min.) DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Concentration of antibody

ANSWER:

Thank you for taking the time to contact us.

I am sorry to hear you have had difficulty obtaining satisfactory results from these antibodies. The details you have kindly provided will enable us to investigate this case for you and also gives us vital information for our monitoring of product quality.

1) When testing our antibodies, our lab uses 5% BSA as a blocking reagent, so I recommend switching to this instead of milk. For unknown reasons 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).  

2) I am unsure if the bands you are observing are around 80 kDa, as the resolution of the higher molecular weight marker bands is suboptimal. In case no bands have run out of the gel and you count from the bottom, the TIMP3 bands could also be around 60 kDa. If that is the case they may represent multimers of the protein (32 kDa dimers were reported according to the literature) or complexes with metalloproteinases. Therefore, I would recommend boiling the samples for more than 5 minutes (e.g. 10min) which may help to reduce disulfide bridges and break up complex structures.

We are happy to offer this technical support. In the event that the product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.  

Madam, Sir,

We recently conducted PCR Array analyses and found five potential targets involved in the response we are assessing. We now want to do Western Blot analyses to confirm the modulation of these five proteins in a lysate of human lung cancer cells. One such protein is interferon alpha 1 for which Abcam offer five different antibodies, i.e. ab 8317, 10077, 86316, 11408 and 37607. We are asking your help to identify the best product for our application. Moreover, we selected the following antibodies for four of these targets and would like you to confirm our choices :

TIMP3 : Ab39184

Maspin : Ab95451

p16 : Ab16123

TIMP1 : Ab61224

Thank you in advance for your technical assistance in our product selection.

Sincerely,

ANSWER:

Thank you for contacting us.

We have indeed in our catalog 5 unconjugated anti-interferon alpha 1 antibodies tested and guaranteed to work in Western Blot and Human samples.

Please note that, unfortunately, import restrictions make the goat polyclonal antibody ab86316 unavailable to Canadian customers.

The remaining 4 products are very similar. However, I would recommend :

- the anti-Interferon alpha antibody clone [AE3] reference ab10077 (www.abcam.com/ab10077) or

- the anti-Interferon alpha antibody clone [C10F5] reference ab8317 (www.abcam.com/ab8317).

These two mouse monoclonal antibodies, protein A purified, are currently in stock, and their unit size is 250µg which makes them cheaper than the other anti-IFNA1.

About the other targets, I agree with you, I would recommend

- ab39184 for the detection of TIMP3,

- ab61224 for TIMP1,

- ab95451 (Mouse monoclonal) for Maspin. ab22354 (Rabbit polyclonal) is also a good choice,

- ab16123 for CDKN2A/p16INK4a. ab54210 is also well characterised with 2 publications and 3 Abreviews (customer feedbacks).

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