Phospho-JNK - (Sep/08/2011 )

Hi ,
I am trying to do western blot for phospho-JNK ,earlier My background was dark & too many bands were there along with phospho bands .
( cell signalling antibody Blocking was in 5% BSA , primary in BSA 1:500 over night , secondry in BSA 1:1000)
then I tried to optimized & got positive control from cell signalling , Changed the method
Block in 5% Milk
primary in BSA 1:1000 overnight
Secondry in BSA 1:2000
Now My blot is very-very clean & got only 2 bands JNK2/JNK1 in positive control,
After thet I tried my samples with positive control ,There was nothing in my sample got bands in only positive control .
Please help me what I am doing wrong ?
Am I losing phosphos all of sudden ?
What should I do ?

-EI0843-

Any one ?

-EI0843-

If am right you have never seen phosphos in your sample, Assuming that your samples are cell lysates, what phosphatase inhibitors you are using in your lysis buffer??
i am using lot of cell signalling abs, it works grt in 5% BSA in TBS, i also block with the same. then for phosphos ofcourse it will work fine with overnight primary, also you can get rid of strong signal by just 1 or 2 hrs primary and some 30 mins blocking...then your phospho probed blots are stripped or virgin? sometimes too strong striping would affect phosphorylation..

-GNANA-

Hi,

My name is Kevin and I work at Cell Signaling Technology. My particular group manages and performs the quality control tests on the SAPK/JNK antibodies. The results from the positive controls indicate that the Western blot and antibody are working properly in your hands. My concern now is that the SAPK/JNK proteins in your samples are either being de-phosphorylated or phosphorylation of SAPK/JNK was not properly induced. Could you provide me with a little more information about your experimental samples? Are they tissue or cell lysates? Were the tissue or cells exposed to some sort of stressor to induce SAPK/JNK phosphorylation? Please feel free to contact me at the email below.

Best,
Kevin

__________________________________
Kevin Tong, Ph.D.
Product Scientist
Cell Signaling Technology
3 Trask Lane
Danvers, MA 01923

Email: kevin.tong@cellsignal.com
Fax: 978-867-2400
WEB Site: www.cellsignal.com

Toll Free:
1-877-616-CELL (2355)
1-877-678-TECH (8324)
Team Extension#: 2391
__________________________________
Please feel free to use our team extension to reach me or a backup
Product Scientist who should also be able to assist you.

-CST Support-

GNANA on Fri Sep 9 08:25:33 2011 said:

If am right you have never seen phosphos in your sample, Assuming that your samples are cell lysates, what phosphatase inhibitors you are using in your lysis buffer??
i am using lot of cell signalling abs, it works grt in 5% BSA in TBS, i also block with the same. then for phosphos of course it will work fine with overnight primary, also you can get rid of strong signal by just 1 or 2 hrs primary and some 30 mins blocking...then your phospho probed blots are stripped or virgin? sometimes too strong striping would affect phosphorylation..

I am adding NAF / NA3VO4 in my RIPA buffer .& Keeping Cells in ice all the time, along with I am running gel at the same day .

-EI0843-

CST Support on Wed Sep 14 13:08:40 2011 said:

I am using INS 832/13 cells & trying to induce P-JNK by treating them with 2.5mM/ 30mM Glucose for 24 hr. .previous lab member have shown beautiful P-JNK using the same method / Lysis buffer (RIPA with NAF & Na3VO4).

-EI0843-

Do you use wet or semid-dry transfer?
for phospho, wet would be much better, with ice, in a cold room.

-little mouse-

If you would not mind, please provide me with a little more information regarding your protocol and lysate preparation by answering the questionnaire below? The information will give me a better handle on your particular situation. My guess is that if your induction is working, it is either not working very well or the phosphate groups are being removed.

Best,
Kevin
__________________________________
Kevin Tong, Ph.D.
Product Scientist
Cell Signaling Technology
3 Trask Lane
Danvers, MA 01923

Email: kevin.tong@cellsignal.com
Fax: 978-867-2400
WEB Site: www.cellsignal.com

Toll Free:
1-877-616-CELL (2355)
1-877-678-TECH (8324)
Team Extension#: 2391
__________________________________
Please feel free to use our team extension to reach me or a backup
Product Scientist who should also be able to assist you.

General Questions

1. Customer Name:

2. Catalog Number:

3. Assay Date printed on tube:

4. Lot Number printed on tube:

5. How long has customer had the product?

6. At what temperature was the product stored?

7. Was the product aliquotted when received? oYes
oNo

Sample Information

1. What cell type was used for the sample lysate? oCell Line
oPrimary Cells
oWhole Tissue
oOther: __________

2. What is the sample species: oHuman
oMouse
oRat
oOther: __________

3. Please state the induction/drug treatment used: ____________________

4. Please state the concentration of the drug used: ____________________

5. Please state the time points of induction: _________________________

6. Please state the positive control used: ____________________________

7. Please select the lysis buffer used: oSDS Sample Buffer
oCST Cell Lysis Buffer
oCST Chaps Buffer
oRIPA Buffer
oOther: ___________

8. What specific phosphatase inhibitors, if any, were included in the lysis buffer?
____________________________________

9. What specific protease inhibitors, if any, were included in the lysis buffer?
____________________________________

10. Were the samples sonicated? o Yes
o No

11. Please select the amount of protein lysate o20 micrograms
loaded per well: oOther: ___________

12. Please state the date of lysate preparation: _____________

13. Please select the lysate storage temperature: o4°C
o-20°C
o-80°C
oOther: ___________

14. Has this antibody previously worked on o Yes
these samples? o No

Protocol Information

1. Please state the % acrylamide in the gel: ___________

2. Please select the membrane used: oNitrocellulose
oPVDF
oNylon
oOther: ___________

Blocking

1. Please state the components of the blocking buffer:__________________
_____________________________________________________________

2. Please state the blocking time and temperature used for blocking step: _____________________

Primary Antibody Incubation

1. Please state the primary antibody source: ______________

2. Please state the primary antibody dilution: ______________

3. Please state the components of the primary antibody dilution buffer: ______
_____________________________________________________________

4. Please state the incubation time and temperature used for the primary antibody incubation: __________________________

5. Please state the washing buffer and time after primary antibody incubation: ________

6. Was the primary antibody dilution re-used or a fresh dilution? ______________

Secondary Antibody Incubation

1. Please state the secondary antibody source: ______________________

2. Please state the secondary antibody dilution: _____________________

3. Please state the components of the secondary antibody dilution buffer: _____________________________________________________________________

4. Please state the incubation time and temperature used for the secondary antibody incubation: __________

5. Please state the washing buffer and time after secondary antibody incubation:
_____________________________________________________________________

Detection

1. Please select the detection system used: oHorseradish Peroxidase (HRP)
oAlkaline Phosphatase (AP)
oOther: ___________________

2. Please state the supplier of the detection system: ____________________________

3. Please state the length of time for film exposure: ____________________________

4. Please describe the appearance of the exposed and developed film. You may attach a scanned image of the blot(s) as a jpg file.

-CST Support-

CST Support on Mon Sep 19 17:42:19 2011 said:

If you would not mind, please provide me with a little more information regarding your protocol and lysate preparation by answering the questionnaire below? The information will give me a better handle on your particular situation. My guess is that if your induction is working, it is either not working very well or the phosphate groups are being removed.

Best,
Kevin
__________________________________
Kevin Tong, Ph.D.
Product Scientist
Cell Signaling Technology
3 Trask Lane
Danvers, MA 01923

Email: kevin.tong@cellsignal.com
Fax: 978-867-2400
WEB Site: www.cellsignal.com

Toll Free:
1-877-616-CELL (2355)
1-877-678-TECH (8324)
Team Extension#: 2391
__________________________________
Please feel free to use our team extension to reach me or a backup
Product Scientist who should also be able to assist you.

General Questions

1. Customer Name:

2. Catalog Number:

3. Assay Date printed on tube:

4. Lot Number printed on tube:

5. How long has customer had the product?

6. At what temperature was the product stored?

7. Was the product aliquotted when received? oYes
oNo

Sample Information

1. What cell type was used for the sample lysate? oCell Line
oPrimary Cells
oWhole Tissue
oOther: ___Cell line_______

2. What is the sample species: oHuman
oMouse
oRat
oOther: ___Rat_______

3. Please state the induction/drug treatment used: _High glucose (30mM)/low glucose(2.5mM)___________________

4. Please state the concentration of the drug used: _30mM/2.5mM___________________

5. Please state the time points of induction: __24 hr._______________________

6. Please state the positive control used: __293 cell treated with UV__________________________

7. Please select the lysis buffer used: oSDS Sample Buffer
oCST Cell Lysis Buffer
oCST Chaps Buffer
oRIPA Buffer
oOther: ___RIPA________

8. What specific phosphatase inhibitors, if any, were included in the lysis buffer?
_NAF/NA3VO4___________________________________

9. What specific protease inhibitors, if any, were included in the lysis buffer?
__Using the Inhibitor cocktail & PMSF__________________________________

10. Were the samples sonicated? NO o Yes
o No

11. Please select the amount of protein lysate o20 micrograms
loaded per well: oOther: __50ug_________

12. Please state the date of lysate preparation: _____________

13. Please select the lysate storage temperature: o4°C
o-20°C
o-80°C
oOther: __NO storage_________

14. Has this antibody previously worked on o Yes
these samples? Yes o No

Protocol Information

1. Please state the % acrylamide in the gel: _10%__________

2. Please select the membrane used: oNitrocellulose
oPVDF
oNylon
oOther: _Nitrocellulose__________

Blocking

1. Please state the components of the blocking buffer:__5% Milk________________
_____________________________________________________________

2. Please state the blocking time and temperature used for blocking step: _RT/ 1hr.____________________

Primary Antibody Incubation

1. Please state the primary antibody source: _Cell signaling_____________

2. Please state the primary antibody dilution: 1:1000/1:500______________

3. Please state the components of the primary antibody dilution buffer: 5%BSA in 1XTBST______
_____________________________________________________________

4. Please state the incubation time and temperature used for the primary antibody incubation: Cold room/Overnight__________________________

5. Please state the washing buffer and time after primary antibody incubation: _3 washes 5min each with 1X TBST_______

6. Was the primary antibody dilution re-used or a fresh dilution? _Fresh_____________

Secondary Antibody Incubation

1. Please state the secondary antibody source: _Millipore_____________________

2. Please state the secondary antibody dilution: _1:2000____________________

3. Please state the components of the secondary antibody dilution buffer: _
5% BSA in 1XTBST____________________________________________________

4. Please state the incubation time and temperature used for the secondary antibody incubation: _1hr. at RT_________

5. Please state the washing buffer and time after secondary antibody incubation:
_______3 washes 5min. each with 1 XTBST______________________________________________________________

Detection

1. Please select the detection system used: oHorseradish Peroxidase (HRP)

oAlkaline Phosphatase (AP)
oOther: _HRP__________________

2. Please state the supplier of the detection system: _Amersham GE healthcare___________________________

3. Please state the length of time for film exposure: _3 min. to 10 min.___________________________

4. Please describe the appearance of the exposed and developed film. You may attach a scanned image of the blot(s) as a jpg file.

-EI0843-

little mouse on Mon Sep 19 15:23:52 2011 said:

Do you use wet or semid-dry transfer?
for phospho, wet would be much better, with ice, in a cold room.

I use wet overnight transfer in cold room.

-EI0843-