Anti-Thrombospondin antibody [A65M] (ab23420)

Our customer had some trouble in trying these antibodies. There are conditions and results below.

We would be appreciated if you could kindly check these questionnaire and give some advices for them. ab23420

1. Order details: a.. Batch number: S060511 b.. Abcam order or Purchase order number: ab23420-100 lot # 220092 c.. Antibody storage conditions (temperature/reconstitution etc) +4°C

2. Please describe the problem (high background, wrong band size, more bands, no band etc). more bands and high background

3. On what material are you testing the antibody in WB? · Species: rat normal liver, Hela and MCF7

· Cell extract or Nuclear extract: cell extract

· Purified protein or Recombinant protein: No

3. The lysate a.. How much protein was loaded: 50 ug a.. What lysis buffer was used: RIPA b.. What protease inhibitors were used: cocktail (roche) c.. What loading buffer was used: sample buffer d.. Did you heat the samples: temperature and time: 95 ?, 5 min

4. Electrophoresis/Gel conditions/ Transfer conditions a.. Reducing or non reducing gel: reducing gel b.. Gel percentage : 8 % c.. Transfer conditions: 400 mA, 2 hr, 4 ?

5. Blocking conditions a.. Buffer: TBST b.. Blocking agent: milk, BSA, serum, what percentage: 5 % milk c.. Incubation time: 1 hr d.. Incubation temperature: 25 ?

6. Primary Antibody a.. Specification (in which species was it raised against): mouse · At what dilution(s) have you tested this antibody: 1: 100

· What dilution buffer was used: 5 % milk in TBST

· Incubation time: 2 hr

· Incubation temperature: 25 ?

· What washing steps were done: washed with TBST 3 times

7. Secondary Antibody a.. Specification (in which species was it raised against)? goat b.. At what dilution(s) have you tested this antibody: 1:5000 c.. Incubation time 1 hr d.. Wash steps: washed with TBST 3 times e.. Do you know whether the problems you are experiencing come from the secondary? yes

8. Detection method ECl, ECl+, other detection method: ECL

9. Background bands · Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): yes

· Is the blocking step sufficient? yes

· Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) yes

· At what size are the bands migrating? Could they be degradation products of your target? no

· Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient)

12% membrane(rat normal liver, Hela and MCF7), expected 128kDa

11. Did you apply positive and negative controls along with the samples? Please specify.

10. Optimization attempts · How many times have you tried the Western? 2 times

· Do you obtain the same results every time e.g. are background bands always in the same place? yes

· What steps have you altered? no

ab22825 PS. When our customer bought this antibody, it was not showed that this is the "Fast-track antibody" last year.

1. Order details: a.. Batch number: S060504 b.. Abcam order or Purchase order number: ab22825 lot:138126 c.. Antibody storage conditions (temperature/reconstitution etc) -20 ?

2. Please describe the problem (high background, wrong band size, more bands, no band etc). more bands and high background

3. On what material are you testing the antibody in WB? · Species:

· Cell extract or Nuclear extract: cell extract

· Purified protein or Recombinant protein: No

3. The lysate a.. How much protein was loaded: 50 ug a.. What lysis buffer was used: RIPA b.. What protease inhibitors were used: cocktail (roche) c.. What loading buffer was used: d.. Did you heat the samples: temperature and time: 95 ?, 5 min

4. Electrophoresis/Gel conditions/ Transfer conditions a.. Reducing or non reducing gel: reducing gel b.. Gel percentage : 20 % c.. Transfer conditions: 400 mA, 2 hr, 4 ?

5. Blocking conditions a.. Buffer: PBST b.. Blocking agent: milk, BSA, serum, what percentage: 5 % milk c.. Incubation time: 1 hr d.. Incubation temperature: 25 ?

6. Primary Antibody a.. Specification (in which species was it raised against): goat · At what dilution(s) have you tested this antibody: 1: 500

· What dilution buffer was used: 5 % milk in PBST

· Incubation time: 2 hr

· Incubation temperature: 25 ?

· What washing steps were done: washed with PBST 3 times

7. Secondary Antibody a.. Specification (in which species was it raised against)? b.. At what dilution(s) have you tested this antibody: 1:5000 c.. Incubation time 1 hr d.. Wash steps: washed with PBST 3 times e.. Do you know whether the problems you are experiencing come from the secondary? yes

8. Detection method ECl, ECl+, other detection method: ECL

9. Background bands · Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): yes

· Is the blocking step sufficient? yes

· Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) yes

· At what size are the bands migrating? Could they be degradation products of your target? no

· Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient)

11. Did you apply positive and negative controls along with the samples? Please specify. 10. Optimization attempts · How many times have you tried the Western? 5 times

· Do you obtain the same results every time e.g. are background bands always in the same place? yes

· What steps have you altered? no

ANSWER:

Thank you for your patience.

I have been in correspondence with the source of each of the antibodies. Unfortunately PTP4A1 antibody (ab22825) is a fast track antibody and has not been applied to a positive control as such.

ab23420 has not been tested by western blotting but has been tested by ELISA against a commercially available source of Thrombospondin from Haematologic Techologies Inc., productnumber: HCTP-0200.

I can tell you that this epitope is only present in thrombospondin prepared with low levels of calcium. The structure is regulated by sulfhydryl-disulfide interchange in the Carboxy-terminal Ca++ binding loops. Indeed the reducing conditions of your customers gel may indeed affect the epitope and potentially be the reason they are not getting the expected results. However, should the antibody work by WB the expected result should be one band at ~150 kDa.

I am sorry I cannot be of more assistance, the lack of antibody characterization makes it difficult.

Our customer had some trouble in trying these antibodies. There are conditions and results below.

We would be appreciated if you could kindly check these questionnaire and give some advices for them. ab23420

1. Order details: a.. Batch number: S060511 b.. Abcam order or Purchase order number: ab23420-100 lot # 220092 c.. Antibody storage conditions (temperature/reconstitution etc) +4°C

2. Please describe the problem (high background, wrong band size, more bands, no band etc). more bands and high background

3. On what material are you testing the antibody in WB? · Species: rat normal liver, Hela and MCF7

· Cell extract or Nuclear extract: cell extract

· Purified protein or Recombinant protein: No

3. The lysate a.. How much protein was loaded: 50 ug a.. What lysis buffer was used: RIPA b.. What protease inhibitors were used: cocktail (roche) c.. What loading buffer was used: sample buffer d.. Did you heat the samples: temperature and time: 95 ?, 5 min

4. Electrophoresis/Gel conditions/ Transfer conditions a.. Reducing or non reducing gel: reducing gel b.. Gel percentage : 8 % c.. Transfer conditions: 400 mA, 2 hr, 4 ?

5. Blocking conditions a.. Buffer: TBST b.. Blocking agent: milk, BSA, serum, what percentage: 5 % milk c.. Incubation time: 1 hr d.. Incubation temperature: 25 ?

6. Primary Antibody a.. Specification (in which species was it raised against): mouse · At what dilution(s) have you tested this antibody: 1: 100

· What dilution buffer was used: 5 % milk in TBST

· Incubation time: 2 hr

· Incubation temperature: 25 ?

· What washing steps were done: washed with TBST 3 times

7. Secondary Antibody a.. Specification (in which species was it raised against)? goat b.. At what dilution(s) have you tested this antibody: 1:5000 c.. Incubation time 1 hr d.. Wash steps: washed with TBST 3 times e.. Do you know whether the problems you are experiencing come from the secondary? yes

8. Detection method ECl, ECl+, other detection method: ECL

9. Background bands · Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): yes

· Is the blocking step sufficient? yes

· Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) yes

· At what size are the bands migrating? Could they be degradation products of your target? no

· Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient)

12% membrane(rat normal liver, Hela and MCF7), expected 128kDa

11. Did you apply positive and negative controls along with the samples? Please specify.

10. Optimization attempts · How many times have you tried the Western? 2 times

· Do you obtain the same results every time e.g. are background bands always in the same place? yes

· What steps have you altered? no

ab22825 PS. When our customer bought this antibody, it was not showed that this is the "Fast-track antibody" last year.

1. Order details: a.. Batch number: S060504 b.. Abcam order or Purchase order number: ab22825 lot:138126 c.. Antibody storage conditions (temperature/reconstitution etc) -20 ?

2. Please describe the problem (high background, wrong band size, more bands, no band etc). more bands and high background

3. On what material are you testing the antibody in WB? · Species:

· Cell extract or Nuclear extract: cell extract

· Purified protein or Recombinant protein: No

3. The lysate a.. How much protein was loaded: 50 ug a.. What lysis buffer was used: RIPA b.. What protease inhibitors were used: cocktail (roche) c.. What loading buffer was used: d.. Did you heat the samples: temperature and time: 95 ?, 5 min

4. Electrophoresis/Gel conditions/ Transfer conditions a.. Reducing or non reducing gel: reducing gel b.. Gel percentage : 20 % c.. Transfer conditions: 400 mA, 2 hr, 4 ?

5. Blocking conditions a.. Buffer: PBST b.. Blocking agent: milk, BSA, serum, what percentage: 5 % milk c.. Incubation time: 1 hr d.. Incubation temperature: 25 ?

6. Primary Antibody a.. Specification (in which species was it raised against): goat · At what dilution(s) have you tested this antibody: 1: 500

· What dilution buffer was used: 5 % milk in PBST

· Incubation time: 2 hr

· Incubation temperature: 25 ?

· What washing steps were done: washed with PBST 3 times

7. Secondary Antibody a.. Specification (in which species was it raised against)? b.. At what dilution(s) have you tested this antibody: 1:5000 c.. Incubation time 1 hr d.. Wash steps: washed with PBST 3 times e.. Do you know whether the problems you are experiencing come from the secondary? yes

8. Detection method ECl, ECl+, other detection method: ECL

9. Background bands · Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): yes

· Is the blocking step sufficient? yes

· Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) yes

· At what size are the bands migrating? Could they be degradation products of your target? no

· Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient)

11. Did you apply positive and negative controls along with the samples? Please specify. 10. Optimization attempts · How many times have you tried the Western? 5 times

· Do you obtain the same results every time e.g. are background bands always in the same place? yes

· What steps have you altered? no

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with this antibody.

PTP4A1 antibody (ab22825) is currently a fast track antibody that we cannot guarantee for the application of western blotting. We appreciate any feedback offered by way of our Abreviews scheme. I would like to direct your customer to www.abcam.com/abreviews. The order that was placed in the past 3 months was sold as a fast track placed. Checking through the records this antibody was purchased in 2005 as a standard price by Gene Research.

The approach that your customer has adopted is very similar to one that I would recommend. I would however like to recommend that they try changing the blocking agent that they have been using to 3% BSA using an overnight incubation and reducing the antibody dilution to 1:50. It would also further assist me if you could send me a blot image of your customers blot so I can better understand the extraneous bands that they have been obtaining.

I am sorry to hear that your customer has also been having difficulties applying Thrombospondin antibody [A65M] (ab23420). This antibody is yet to be applied using western blotting and therefore we cannot guarantee it for this purpose. However, I would recommend performing an approach akin to the one above; using BSA and reducing the dilution of the antibody.

I am in touch with the originators of both antibodies in order to source information as to the most suitable positive control as I am not confident that HeLa and MCF7 cells are the best option. I will be in touch shortly. I appreciate your patience.

I have a question of clarification regarding the uses of this product. The indications include "IHC" as well as "IHC-F". I need to use this antobody for IHC on parafin-fixed specimens, and I know you usually have an indication specifically for this. I wanted to clarify if this antibody can be used for such a process.

ANSWER:

Thank you for your enquiry. Ab23420 has only so far been tested for application in IHC-frozen tissue sections. It has not yet been tested with paraffin embedded sections.

If you decide to go ahead and purchase this product, please let us know how you get on by submitting an Abreview and in return we will award you 50 Abcam Points, which can be redeemed on a number of rewards (a further 100 Abcam Points will be offered for an image).

Please contact us again if you have any additional questions.