Overview

Properties

Applications

Our Abpromise guarantee covers the use of ab3469 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-P Use at an assay dependent concentration.
WB Use a concentration of 10 mg/ml. Detects a band of approximately 52 kDa (predicted molecular weight: 36 kDa).

By Western blot, this antibody detects an ~52 kDa protein representing Fra 2 from TPA stimulated HeLa cell lysate. The band at ~52 kDa is thought to represent the phosphorylated form of Fra2.

Target

References

ab3469 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question

BATCH NUMBER lot 9198 ORDER NUMBER P0131A DESCRIPTION OF THE PROBLEM Multiple bands. Major bands detected at >200k, 70k and 50k. Six other bands appear on longer exposure (150 to 50k). SAMPLE HeLa nuclear extract. Similar results with Jurkat and 70 Z/3. PRIMARY ANTIBODY AbCam ab 3469 at 10 or 5 ug/mL (1:100 or 1:200) in 1% NFDM/Tween/TBS, O/N at 4 C on rocker. SECONDARY ANTIBODY Southern Biotech goat anti-rabbit at 1:2000 in 1% NFDM/Tween/TBS, 60 or 90 minutes, RT on rocker. DETECTION METHOD Roche Lumi-Light. POSITIVE AND NEGATIVE CONTROLS USED No positive control. ANTIBODY STORAGE CONDITIONS Reconstituted lyophilised product at 1 mg/mL with water. Stored at 4 C. SAMPLE PREPARATION Extracts prepared by Dignam method. Dialyzed against .5 mM PMSF before frozen storage. AMOUNT OF PROTEIN LOADED Tried 20 and 10 ug protein/lane. ELECTROPHORESIS/GEL CONDITIONS 7% or 10% acrylamide gels. Samples boiled with 2-ME before loading. Reductants not included in gel or buffers. TRANSFER AND BLOCKING CONDITIONS Transfer 20 or 60 minutes in glycine/tris/methanol at 100 V. Prestained size markers included on gel, so efficient transfer was verified. Blocked one hour in 5% NFDM/Tween/TBS. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Changed sample size, transfer time, primary antibody concentration and secondary antibody incubation time. ADDITIONAL NOTES Target presence in sample was verified by mass spec in a 35k PAGE band. Theoretical protein mass is 35k. Santa Cruz catalog (p97) shows detection of Fra2 at about 40k. We see no significant signal below 48k. We don't need to eliminate the inappropriate signals, if we can identify the true signal.

Read More
Answer

Thank you for taking the time to submit this complaint. We have looked through the protocol that you have forwarded to us, as well as seeking additional information from the originator of the product and seeking the protocol that was used to test this Ab. There are some suggestions that may be made that may help with the results that you are gaining. 1) Incubate the primary Ab for 2 hrs at room temperature and reduce the concentration that you are using the primary Ab at. Fra2 shows a band of approximately 50KDa using this Ab in HeLa cells. This KDa is suspected to be the molecular weight of the phophorylated form of the target, from your description it sounds as though the band of interest is present. 2) From the information that you have forwarded you did not add reducing agents to the gel or buffers. The blot was initially run using gels containing B-mercaptoethanol and SDS on a 8% gel with a 5% attacking gel. 3) Normally I would suggest using additional protease inhibitors leupeptin and pepstatin in addition to PMSF to prevent target breakdown (particularly if you are seeing bands below the expected target size). 4) When initially testing this Ab the wash steps used were 6 changes over the course of one 1hr in PBS/T with vigorous shaking. 5) Is there any non specific binding observed when the no primary control is run? 6) Test Sample Preparation. The cells for testing this Ab were snap frozen before storage, this may be an important step. Total cellular proteins were isolated by suspending the cells in 10 vol of 1x Laemmli sample buffer (0.062 M Tris/HCl, pH 6.8, 5% glycerol, 2% SDS) and then sonicating the cell lysate on ice. Protein was estimated using a Bio-Rad DC Protein Assay Kit. Lysates were flash frozen in liquid nitrogen and stored at –80 °C. 7) When testing this Ab HeLa cells were stimulated with 1x10-7 M TPA stimulated (2h). You could also run your non stimulated samples against samples stimulated with TPA. The originator of the product states that they received one clean band in HeLa cells. If you are still experiencing trouble then please get back in touch with me. I have cut and pasted the protocol used to test this product below. "Test Sample Preparation: Total cellular proteins were isolated by suspending the cells in 10 vol of 1x Laemmli sample buffer (0.062 M Tris/HCl, pH 6.8, 5% glycerol, 2% SDS) and then sonicating the cell lysate on ice. Protein was estimated using a Bio-Rad DC Protein Assay Kit. Lysates were flash frozen in liquid nitrogen and stored at –80 °C. Gel Conditions: ß-Mercaptoethanol and bromophenol blue tracking dye was added to a final concentration of 1% and 5 ug/ml, respectively. Total cellular protein (20 ug/lane) was separated by discontinuous SDS PAGE (mini gels) using an 8% separating gel and a 5% stacking gel. Transfer Conditions: Proteins were transferred to nitrocellulose (BA 85, S & S) using a Gelman semidry transfer unit (Transfer buffer: Tris-base 48 mM, glycine-HCl 39 mM, SDS 1.3 mM, methanol 20%, pH 9.2) and stained with Ponceau S to confirm uniformity of transfer. Blocking Solution & Duration: 5% non fat dry milk and 0.02% merthiolate in PBS (phosphate-buffered saline), overnight at 4 °C, gentle shaking. Primary Antibody Incubation Time & Temperature: 2 h at room temperature. Wash Solution Composition, Repetitions & Time: Washed in PBS/T, 6 changes over 1 h, vigorous shaking. Secondary Antibody: Used HRP-labelled goat anti-rabbit IgG (H + L) 1:5000 Secondary Antibody Incubation Time and Temperature: 1 h at room temperature. Wash Solution Composition, Repetitions & Times: Washed in PBS/T, 6 changes over 1 h, vigorous shaking. Detection system Amersham ECL kit. Used Amersham Hyperfilm-MP. Molecular weight of band that is seen: Reported apparent molecular weight ~46 kDa. There is no Fra2 specific signal when using NIH 3T3 cell extracts. There appears to be a Fra2 specific band at ~52 kDa when using HeLa cell extracts. This is the only protein detected by this Ab, and due to its slower migration, it could represent a phosphorylated form of Fra2. Species, Tissues and Cell Lines Tested: 20% Serum stimulated (2h) mouse NIH 3t3 and 1x10-7 M TPA stimulated (2h) human HeLa cells. Conclusions and Impressions: Supershift analysis 10/20/95 showed that Fra2 was unable to supershift protein/DNA complexes formed when using NIH 3T3 or HeLa cell nuclear extracts and AP-1 sites. There appears to be a Fra2 specific band at ~52 kDa. This is the only protein detected by this Ab and represents a phosphorylated form of Fra2".

Read More

Question

BATCH NUMBER lot 9198 ORDER NUMBER P0131A DESCRIPTION OF THE PROBLEM Multiple bands. Major bands detected at >200k, 70k and 50k. Six other bands appear on longer exposure (150 to 50k). SAMPLE HeLa nuclear extract. Similar results with Jurkat and 70 Z/3. PRIMARY ANTIBODY AbCam ab 3469 at 10 or 5 ug/mL (1:100 or 1:200) in 1% NFDM/Tween/TBS, O/N at 4 C on rocker. SECONDARY ANTIBODY Southern Biotech goat anti-rabbit at 1:2000 in 1% NFDM/Tween/TBS, 60 or 90 minutes, RT on rocker. DETECTION METHOD Roche Lumi-Light. POSITIVE AND NEGATIVE CONTROLS USED No positive control. ANTIBODY STORAGE CONDITIONS Reconstituted lyophilised product at 1 mg/mL with water. Stored at 4 C. SAMPLE PREPARATION Extracts prepared by Dignam method. Dialyzed against .5 mM PMSF before frozen storage. AMOUNT OF PROTEIN LOADED Tried 20 and 10 ug protein/lane. ELECTROPHORESIS/GEL CONDITIONS 7% or 10% acrylamide gels. Samples boiled with 2-ME before loading. Reductants not included in gel or buffers. TRANSFER AND BLOCKING CONDITIONS Transfer 20 or 60 minutes in glycine/tris/methanol at 100 V. Prestained size markers included on gel, so efficient transfer was verified. Blocked one hour in 5% NFDM/Tween/TBS. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Changed sample size, transfer time, primary antibody concentration and secondary antibody incubation time. ADDITIONAL NOTES Target presence in sample was verified by mass spec in a 35k PAGE band. Theoretical protein mass is 35k. Santa Cruz catalog (p97) shows detection of Fra2 at about 40k. We see no significant signal below 48k. We don't need to eliminate the inappropriate signals, if we can identify the true signal.

Read More
Answer

Thank you for taking the time to submit this complaint. We have looked through the protocol that you have forwarded to us, as well as seeking additional information from the originator of the product and seeking the protocol that was used to test this Ab. There are some suggestions that may be made that may help with the results that you are gaining. 1) Incubate the primary Ab for 2 hrs at room temperature and reduce the concentration that you are using the primary Ab at. Fra2 shows a band of approximately 50KDa using this Ab in HeLa cells. This KDa is suspected to be the molecular weight of the phophorylated form of the target, from your description it sounds as though the band of interest is present. 2) From the information that you have forwarded you did not add reducing agents to the gel or buffers. The blot was initially run using gels containing B-mercaptoethanol and SDS on a 8% gel with a 5% attacking gel. 3) Normally I would suggest using additional protease inhibitors leupeptin and pepstatin in addition to PMSF to prevent target breakdown (particularly if you are seeing bands below the expected target size). 4) When initially testing this Ab the wash steps used were 6 changes over the course of one 1hr in PBS/T with vigorous shaking. 5) Is there any non specific binding observed when the no primary control is run? 6) Test Sample Preparation. The cells for testing this Ab were snap frozen before storage, this may be an important step. Total cellular proteins were isolated by suspending the cells in 10 vol of 1x Laemmli sample buffer (0.062 M Tris/HCl, pH 6.8, 5% glycerol, 2% SDS) and then sonicating the cell lysate on ice. Protein was estimated using a Bio-Rad DC Protein Assay Kit. Lysates were flash frozen in liquid nitrogen and stored at –80 °C. 7) When testing this Ab HeLa cells were stimulated with 1x10-7 M TPA stimulated (2h). You could also run your non stimulated samples against samples stimulated with TPA. The originator of the product states that they received one clean band in HeLa cells. If you are still experiencing trouble then please get back in touch with me. I have cut and pasted the protocol used to test this product below. "Test Sample Preparation: Total cellular proteins were isolated by suspending the cells in 10 vol of 1x Laemmli sample buffer (0.062 M Tris/HCl, pH 6.8, 5% glycerol, 2% SDS) and then sonicating the cell lysate on ice. Protein was estimated using a Bio-Rad DC Protein Assay Kit. Lysates were flash frozen in liquid nitrogen and stored at –80 °C. Gel Conditions: ß-Mercaptoethanol and bromophenol blue tracking dye was added to a final concentration of 1% and 5 ug/ml, respectively. Total cellular protein (20 ug/lane) was separated by discontinuous SDS PAGE (mini gels) using an 8% separating gel and a 5% stacking gel. Transfer Conditions: Proteins were transferred to nitrocellulose (BA 85, S & S) using a Gelman semidry transfer unit (Transfer buffer: Tris-base 48 mM, glycine-HCl 39 mM, SDS 1.3 mM, methanol 20%, pH 9.2) and stained with Ponceau S to confirm uniformity of transfer. Blocking Solution & Duration: 5% non fat dry milk and 0.02% merthiolate in PBS (phosphate-buffered saline), overnight at 4 °C, gentle shaking. Primary Antibody Incubation Time & Temperature: 2 h at room temperature. Wash Solution Composition, Repetitions & Time: Washed in PBS/T, 6 changes over 1 h, vigorous shaking. Secondary Antibody: Used HRP-labelled goat anti-rabbit IgG (H + L) 1:5000 Secondary Antibody Incubation Time and Temperature: 1 h at room temperature. Wash Solution Composition, Repetitions & Times: Washed in PBS/T, 6 changes over 1 h, vigorous shaking. Detection system Amersham ECL kit. Used Amersham Hyperfilm-MP. Molecular weight of band that is seen: Reported apparent molecular weight ~46 kDa. There is no Fra2 specific signal when using NIH 3T3 cell extracts. There appears to be a Fra2 specific band at ~52 kDa when using HeLa cell extracts. This is the only protein detected by this Ab, and due to its slower migration, it could represent a phosphorylated form of Fra2. Species, Tissues and Cell Lines Tested: 20% Serum stimulated (2h) mouse NIH 3t3 and 1x10-7 M TPA stimulated (2h) human HeLa cells. Conclusions and Impressions: Supershift analysis 10/20/95 showed that Fra2 was unable to supershift protein/DNA complexes formed when using NIH 3T3 or HeLa cell nuclear extracts and AP-1 sites. There appears to be a Fra2 specific band at ~52 kDa. This is the only protein detected by this Ab and represents a phosphorylated form of Fra2".

Read More

Question

BATCH NUMBER lot 9198 ORDER NUMBER P0131A DESCRIPTION OF THE PROBLEM Multiple bands. Major bands detected at >200k, 70k and 50k. Six other bands appear on longer exposure (150 to 50k). SAMPLE HeLa nuclear extract. Similar results with Jurkat and 70 Z/3. PRIMARY ANTIBODY AbCam ab 3469 at 10 or 5 ug/mL (1:100 or 1:200) in 1% NFDM/Tween/TBS, O/N at 4 C on rocker. SECONDARY ANTIBODY Southern Biotech goat anti-rabbit at 1:2000 in 1% NFDM/Tween/TBS, 60 or 90 minutes, RT on rocker. DETECTION METHOD Roche Lumi-Light. POSITIVE AND NEGATIVE CONTROLS USED No positive control. ANTIBODY STORAGE CONDITIONS Reconstituted lyophilised product at 1 mg/mL with water. Stored at 4 C. SAMPLE PREPARATION Extracts prepared by Dignam method. Dialyzed against .5 mM PMSF before frozen storage. AMOUNT OF PROTEIN LOADED Tried 20 and 10 ug protein/lane. ELECTROPHORESIS/GEL CONDITIONS 7% or 10% acrylamide gels. Samples boiled with 2-ME before loading. Reductants not included in gel or buffers. TRANSFER AND BLOCKING CONDITIONS Transfer 20 or 60 minutes in glycine/tris/methanol at 100 V. Prestained size markers included on gel, so efficient transfer was verified. Blocked one hour in 5% NFDM/Tween/TBS. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Changed sample size, transfer time, primary antibody concentration and secondary antibody incubation time. ADDITIONAL NOTES Target presence in sample was verified by mass spec in a 35k PAGE band. Theoretical protein mass is 35k. Santa Cruz catalog (p97) shows detection of Fra2 at about 40k. We see no significant signal below 48k. We don't need to eliminate the inappropriate signals, if we can identify the true signal.

Read More
Answer

Thank you for taking the time to submit this complaint. We have looked through the protocol that you have forwarded to us, as well as seeking additional information from the originator of the product and seeking the protocol that was used to test this Ab. There are some suggestions that may be made that may help with the results that you are gaining. 1) Incubate the primary Ab for 2 hrs at room temperature and reduce the concentration that you are using the primary Ab at. Fra2 shows a band of approximately 50KDa using this Ab in HeLa cells. This KDa is suspected to be the molecular weight of the phophorylated form of the target, from your description it sounds as though the band of interest is present. 2) From the information that you have forwarded you did not add reducing agents to the gel or buffers. The blot was initially run using gels containing B-mercaptoethanol and SDS on a 8% gel with a 5% attacking gel. 3) Normally I would suggest using additional protease inhibitors leupeptin and pepstatin in addition to PMSF to prevent target breakdown (particularly if you are seeing bands below the expected target size). 4) When initially testing this Ab the wash steps used were 6 changes over the course of one 1hr in PBS/T with vigorous shaking. 5) Is there any non specific binding observed when the no primary control is run? 6) Test Sample Preparation. The cells for testing this Ab were snap frozen before storage, this may be an important step. Total cellular proteins were isolated by suspending the cells in 10 vol of 1x Laemmli sample buffer (0.062 M Tris/HCl, pH 6.8, 5% glycerol, 2% SDS) and then sonicating the cell lysate on ice. Protein was estimated using a Bio-Rad DC Protein Assay Kit. Lysates were flash frozen in liquid nitrogen and stored at –80 °C. 7) When testing this Ab HeLa cells were stimulated with 1x10-7 M TPA stimulated (2h). You could also run your non stimulated samples against samples stimulated with TPA. The originator of the product states that they received one clean band in HeLa cells. If you are still experiencing trouble then please get back in touch with me. I have cut and pasted the protocol used to test this product below. "Test Sample Preparation: Total cellular proteins were isolated by suspending the cells in 10 vol of 1x Laemmli sample buffer (0.062 M Tris/HCl, pH 6.8, 5% glycerol, 2% SDS) and then sonicating the cell lysate on ice. Protein was estimated using a Bio-Rad DC Protein Assay Kit. Lysates were flash frozen in liquid nitrogen and stored at –80 °C. Gel Conditions: ß-Mercaptoethanol and bromophenol blue tracking dye was added to a final concentration of 1% and 5 ug/ml, respectively. Total cellular protein (20 ug/lane) was separated by discontinuous SDS PAGE (mini gels) using an 8% separating gel and a 5% stacking gel. Transfer Conditions: Proteins were transferred to nitrocellulose (BA 85, S & S) using a Gelman semidry transfer unit (Transfer buffer: Tris-base 48 mM, glycine-HCl 39 mM, SDS 1.3 mM, methanol 20%, pH 9.2) and stained with Ponceau S to confirm uniformity of transfer. Blocking Solution & Duration: 5% non fat dry milk and 0.02% merthiolate in PBS (phosphate-buffered saline), overnight at 4 °C, gentle shaking. Primary Antibody Incubation Time & Temperature: 2 h at room temperature. Wash Solution Composition, Repetitions & Time: Washed in PBS/T, 6 changes over 1 h, vigorous shaking. Secondary Antibody: Used HRP-labelled goat anti-rabbit IgG (H + L) 1:5000 Secondary Antibody Incubation Time and Temperature: 1 h at room temperature. Wash Solution Composition, Repetitions & Times: Washed in PBS/T, 6 changes over 1 h, vigorous shaking. Detection system Amersham ECL kit. Used Amersham Hyperfilm-MP. Molecular weight of band that is seen: Reported apparent molecular weight ~46 kDa. There is no Fra2 specific signal when using NIH 3T3 cell extracts. There appears to be a Fra2 specific band at ~52 kDa when using HeLa cell extracts. This is the only protein detected by this Ab, and due to its slower migration, it could represent a phosphorylated form of Fra2. Species, Tissues and Cell Lines Tested: 20% Serum stimulated (2h) mouse NIH 3t3 and 1x10-7 M TPA stimulated (2h) human HeLa cells. Conclusions and Impressions: Supershift analysis 10/20/95 showed that Fra2 was unable to supershift protein/DNA complexes formed when using NIH 3T3 or HeLa cell nuclear extracts and AP-1 sites. There appears to be a Fra2 specific band at ~52 kDa. This is the only protein detected by this Ab and represents a phosphorylated form of Fra2".

Read More

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Sign up