Antibody Verify Holiday Hours-November 2013

The Antibody Verify office will be closed for Thanksgiving, Thursday November 28th and Friday November 29th. Shipping times will be affected this week.

Monday, November 25th: Regular Office hours, shipping all orders.
Tuesday, November 26th: Regular Office hours. Only domestic shipments and International shipments not on ice processed.
Wednesday, November 27th: Regular Office hours. Only International and Canadian shipments will be processed.
Monday, December 2nd: Regular Office Hours. Regular shipping schedules will resume.

**Please note: International shipments on ice will only be shipped on Monday, November 25th. If orders are not received by Friday November 22nd, they may be held until Monday, December 2nd.

Thank you.

The Antibody Verify Team

Hundreds of Immunohistochemistry Experiments Submitted to Antibody Verify

Although immunohistochemistry (IHC)has been used in proteomics for decades, it still is one of our most popular pieces of data submitted by researchers. To view a list of IHC customer reviews, please click here.

Looking To Test Many Antibodies?

Antibody Verify can assist you with large projects by greatly reducing the cost of antibodies. If you have a large project and would like us to help, please send us an email to info@antibodyverify.com with the following details:

  • List of targets and species
  • Accession numbers if available
  • Amount of antibody needed
  • Application and sample types to be tested

Send Us A Request

Antibody Verify has a network of antibody manufacturers who are seeking to produce antibodies for researchers in exchange for validation data. To have us make an antibody for you, please send the following details to info@antibodyverify.com:

  • Target name and species
  • Protein region of interest to detect
  • Application for antibodies use
  • Desired host species

Product Review: TAZ Antibody (AAS99633C) in mouse cardiac mitochondria lysate using Western blot

Product Page for TAZ Antibody-C-terminal region (AAS99633C)

Researcher:Corey Powers, Cincinati Children’s Hospital Medical Center
Application:Western blotting
Species+tissue/cell type:
Lane 1: 20ug mouse cardiac mitochondria lysate
Lane 2: 20ug mouse cardiac mitochondria lysate
Lane 3: 20ug mouse cardiac mitochondria lysate
Lane 4: 20ug mouse cardiac mitochondria lysate
Primary antibody dilution:1:500
Secondary antibody:Goat anti-rabbit-HRP
Secondary antibody dilution:1:2500

Questionnaire:
How do Antibody Verify’s reagents play a role in your experimental goals? Tafazzin is a target gene in our research, thus a solid antibody would enbale further analysis for us.
Would you use this antibody in future experiments? If was improved, yes.
If the antibody works, do you plan to use it in future experiments or to publish your data? Why or why not? We would like to use it, however more antibody would be required to validate the spotty results that I have gotten.
How did you store the antibody after re-suspension?  -20C in aliquots
Sample Description (please include 1) species type, and 2) cell/tissue type, and 3) how much protein loaded for each lane of your gel): 1) Mus musculus 2) Cardiac mitochondria 3) 20ug protein/Lane
How many different experimental trials were conducted using the antibody sample? 4 trials
How was this sample prepared? Sample was lysed with KH2PO4 + 2% Triton X-100. Then added H20, loading buffer and a reducing agent. Then heated to 95C for 10 minutes prior to loading.
Primary antibody dilution and incubation time: 1:500 in 5% BSA + PBS-T, Overnight at 4C with a agitation.
Secondary antibody used and dilution and incubation time: Goat Anti-Rabbit-HRP 1:2500, 1 Hour at RT with 5% milk + PBS-T
What controls were used in your experiment (positive/negative)? We used a + Ab control, mMDH which shows up in all of our samples.
Please include your detailed WB Procedure/Protocol here: Lysed with KH2PO4 + 2% Triton X-100, incubate sample on ice 30 min.
Add 4X loading buffer, 10X Reducing agent, H2O and sample to 20ul total volume. Protein was added to ensure 20ug in each lane (40ug total).
Sample were heated to 95C for 10 minutes and then iced until loading. We use the invitrogen Novex system. Thus we use a 1x MOPS running buffer and 1x MOPS in the inner chamber of the gel chamber with 500ul of Antioxidant. We used a 4-12% Bis-Tris Gel. The gel was run at 120V for ~2 hours. We then used PVDF membrane and a semi-dry transfer unit to transfer for 30 minutes at 25V. We washed the membrane in 0.5% PBS-Tween20 3×15 minutes and then blocked overnight at 4C in 5% BSA+PBS-T. We then washed 3×15 minutes in PBS-T. Wash 3×15 minutes w/ PBS-T, incubate for 1 Hour with secondary Goat-Anti-Rabbit HRP 1:2500 in 5% milk + PBS-T. Wash 3×15 minutes with PBS-T and then detect using a GE STORM with GE ECL Prime with a 1:1 ratio of the reagents for a total of 1ml.

Product Review: COL1A1 Antibody (AAS99599C) in Mouse blastocyte using IHC

Product Page for COL1A1 antibody-C-terminal region (AAS99599C)

Researcher: Ivan Bedzhov, PhD, Gurdon Institute, University of Cambridge
Application: IHC
Species+tissue/cell type:Species+tissue/cell type: Mouse blastocyte
Primary antibody dilution: 1:200
Secondary antibody: Anti-rabbit-Alexa 488
Secondary antibody dilution:1:200


Questionnaire: How do Antibody Verify’s reagents play a role in your experimental goals? How would you rate this antibody on a scale from 1-5 (5=best) and why? 5, clear and shARP signal. Would you use this antibody in future experiments? Yes Have you used another antibody which has worked in your application? Yes Do you believe the information about the reagent on Antibody Verify’s website is correct? Yes If the antibody works, do you plan to use it in future experiments or to publish your data? Why or why not? Yes, I plan to use it and evantually publish. How did you store the antibody after re-suspension? 4 degress Please explain fixation solution/method used (formalin, periodate-lysine-paraformaldehyde, acetone, etc.)? PFA How many different experimental trials were conducted using the antibody sample? 3 Primary antibody dilution, incubation time and temperature: At 1:200 Secondary antibody used, dilution, incubation time and temperature: Alexa488 From your IHC/ICC images, briefly explain the colors of each stain and counterstain: Green: Collagen, Blue: DAPI Did you use an antigen retrieval method? If so, please explain? No What controls were used in your experiment? Please include your positive control: Blastocyst outgrowth stained for Laminin Experimental Procedure/Protocols: Fix in 2% PFA in PBS.
Wash 3X PBS
Permeabilize 0.5% Triton-X-100 in PBS, 15 min
Wash 2X PBS
Primary antibody 1:200 in PBS, overnight, 4 degrees
Wash 3X PBS
Secondary antibody 1:200 in PBS, overnight, 4 degress or 2h RT
Wash 3X PBS

Product Review: Sdpr Antibody (AAS99504C) in Human placental and myometrial tissue lysate using Western Blot

Product Page for Sdpr antibody-N-terminal region (AAS99504C)

Researcher: Hiten Mistry, Ania Czajka and Marta Hentschke Ribeiro, King’s College London
Application: Western blotting
Species+tissue/cell type: Human placental and myometrial tissue lysate
How many ug’s of tissue/cell lysate run on the gel:
1: 30 ug human placental tissue lysate
2: 30 ug human placental tissue lysate
3: 30 ug human placental tissue lysate
4: 30 ug human placental tissue lysate
5: 20 ug human myometrial tissue lysate
Primary antibody dilution: 1:500
Secondary antibody: Goat anti-rabbit HRP
Secondary antibody dilution: 1:10000

Questionnaire:

How do Antibody Verify’s reagents play a role in your experimental goals?

It is one of our main antibodies of interest.

How would you rate this antibody on a scale from 1-5 (5=best) and why?

4. It did worked for tissue lysates of interest and for our positive control.

Would you use this antibody in future experiments?

Yes

Have you used another antibody which has worked in your application?

No

Do you believe the information about the reagent on Antibody Verify’s website is correct?

Yes

If the antibody works, do you plan to use it in future experiments or to publish your data? Why or why not?

Yes

How did you store the antibody after re-suspension?

-20C

Sample Description (please include 1) species type, and 2) cell/tissue type, and 3) how much protein loaded for each lane of your gel):

1. Human, 2. Placental lysate 3. 20-50ug/lane

How many different experimental trials were conducted using the antibody sample?

2-3

How was this sample prepared?

100mg of sample was homogenised in RIPA buffer and treated with protease inhibitors cocktail (SIGMA).

Primary antibody dilution and incubation time:

1:500, overnight incubation

Secondary antibody used and dilution and incubation time:

1:10000, 2-3 hrs

What controls were used in your experiment (positive/negative)?

Positive: myometrial tissue lysate

Please include your detailed WB Procedure/Protocol here:

Equipment: XCell SureLock Mini-cell system with blotting module, gel loading, pipette, tips, mechanical rocker.

1. Loading buffer: Laemlli buffer (Protein loaded (u/5) required to run for each sample)

2. Running buffer (MES SDS RB 20X)

3.Marker (10ug of Novex 4-12% Bis-Tris Gel 1X Mes Running buffer)

4. Gel: 4-12% Bis-Tris gel

5. Electrophorosis Conditions: 1h-1h30min, 175V

6. Transfer Membrane used: Immobilion-P (Millipore) (PVDF)

7. Block in methanol X2 (30 sec/15 sec)

8. Blotting conditions: 1h 30 min, 30V

9. Primary antibody (1:500), overnight Incubation

10. Wash 3 times for 20 minutes at maximum agitation in TBS-Tween after antibodies incubation

11. ECL (3mL per membrane, equal quantities of reagents A and B) for 1 minute

12. Blot analysis: Chemiluminescense (ECL TM)

13. Visualization useing GelLogic 2200 Pro, Carestream Program, from 10sec -30 mindepending on antibody

Product Review: NOVA2 Antibody (AAS99403E) in human neural cancer cell line using Western Blot

Product Page for NOVA2 antibody – N-terminal region (AAS99403E)

Researcher: Dr. Monica Faronato, University of Liverpool
Application: Western blotting
Species+tissue/cell type: Human neural cell line known to express NOVA2
How many ug’s of tissue/cell lysate run on the gel:  10 ug human neural cell lysate
Primary antibody dilution: 1:1000
Secondary antibody: Goat anti-rabbit IRDye 800
Secondary antibody dilution: 1:15,000

Questionnaire:

How do Antibody Verify’s reagents play a role in your experimental goals?

Determining relative expression across cell lines .

How would you rate this antibody on a scale from 1-5 (5=best) and why?

5.  Very good clean signal by immunoblotting.

Would you use this antibody in future experiments?

Yes.

Have you used another antibody which has worked in your application?

No.

Do you believe the information about the reagent on Antibody Verify’s website is correct?

Yes.

If the antibody works, do you plan to use it in future experiments or to publish your data? Why or why not?

Yes.

Sample Description (please include 1) species type, and 2) cell/tissue type, and 3) how much protein loaded for each lane of your gel):

Human. different types of normal and cancer cells. 10ug.

How many different experimental trials were conducted using the antibody sample?

3

What type of experimental sample are you using and how did you preparing it?

Whole cell lysate prepared in Laemmli buffer (2% SDS, 50mM Tris pH 6.8,10% glycerol).

Primary used and dilution:

1 in 1000, overnight incubation at 4°C.

Secondary used and dilution:

Goat anti-rabbit IRDye 800, 1 in 15,000, 1hr room temperature.

Experimental Procedure/Protocols:

‘Cell lysates were prepared by fractionation or by whole cell lysis:

Whole cell lysis: samples were lysed directly on heat block at 110 degrees in Laemmli buffer (2% SDS, 50mM Tris pH 6.8,10% glycerol)

The following amounts of extracts were used for immunoblotting: 10µg whole cell extract.

(*) Blocking buffer: 5% non-fat dry milk in 1X PBS, pH=7.4

(**) Antibody dilution buffer: 5% non-fat dry milk 0.05% PBST

(***) Membrane washing buffer 0.1% PBST

Membranes were blocked in blocking buffer (*) for 1 hour at room temperature.

Membrane pre-treatment: one wash in distilled water for 20 minutes.

The primary antibodies were diluted 1:500 in antibody dilution buffer (**) and incubated overnight with the membranes (in a cold room at 4°C with agitation to ensure homogenous coverage).

The following day, the primary antibody was poured off and the membranes washed 6 times for 5 minutes each time in membrane washing buffer (***).

Secondary antibody (IRDye 800 conjugated) was diluted in (**) and incubated for 1 hour at room temperature.

Secondary antibody was poured off and the membrane washed 6 times for 5 minutes each time in (***).

A Licor Odyssey system was used to image immunoblots.’

What controls were used in your experiment? Please include your positive control:

Cells known to express or not express NOVA2.

How did you store the antibody after re-suspension?

At -20°C in small working aliquots.

Product Review: GJA1 Antibody (AAS98365C) in rat cardiac lysate using Western Blot

Product Page for GJA1 antibody-N-terminal region (AAS98365C)

Application: Western blotting
Species+tissue/cell type: Total rat cardiac lysate
How many ug’s of tissue/cell lysate run on the gel:
1: 8 ug total cardiac lysate
2: 15 ug total cardiac lysate
3: 30 ug total cardiac lysate
4: 50 ug total cardiac lysate
Primary antibody dilution: 0.25 ug/ml
Secondary antibody:
Secondary antibody dilution:

Product Review: ATP7A Antibody (AAS98337C) in mouse endothelial firboblast and human HUVEC using Western blot

Product Page for ATP7A antibody-middle region (AAS98337C)

Researcher:Anonymous
Application: Western blotting
Species+tissue/cell type:Lane1: 40 ug mouse endothelial firboblast lysate, Lane2: 40 ug human HUVEC lysate
Primary antibody dilution: 1:500
Secondary antibody: Anti-rabbit HRP
Secondary antibody dilution: 1:2000

Questionnaire:
How would you rate this antibody on a scale from 1-5 (5=best) and why? 5- for IB
Would you use this antibody in future experiments? YES
Have you used another antibody which has worked in your application? YES
Do you believe the information about the reagent on Antibody Verify’s website is correct? YES
If the antibody works, do you plan to use it in future experiments or to publish your data? Why or why not? YES
How did you store the antibody after re-suspension? Aliquote in different tube and stored in -20 C
Sample Description (please include 1) species type, and 2) cell/tissue type, and 3) how much protein loaded for each lane of your gel): 1)Mouse, Human 2)fibroblast cells, Endothelial cells, 40 ug protein
How many different experimental trials were conducted using the antibody sample? 1
How was this sample prepared? Cells were lysed with 500 ?l of ice-cold lysis buffer, pH 7.4 (50 mM HEPES, 5 mM EDTA, 150 mM NaCl), 1% Triton X-100, protease inhibitors (10 ?g/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 10 ?g/ml leupeptin).
Primary antibody dilution and incubation time: 1:500, overnight
Secondary antibody used and dilution and incubation time: Anti-Rabbit, 1:2000, 1 hr
What controls were used in your experiment (positive/negative)? Positive
Please include your detailed WB Procedure/Protocol here: I followed the instruction for IB from Antibody Verify website .