Anti-Lamin B1 antibody [119D5-F1] - Nuclear Envelope Marker (ab8982)

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 102450

DESCRIPTION OF THE PROBLEM No Signal at around 70kD. However, weird smaller MW bands appear after long exposure of the film.

SAMPLE NRK nuclear extract

PRIMARY ANTIBODY I used 1:100 dilution into I-block. The second time I tried 1:50 dilution. I incubated for 1 hour at least at room temperature, then three washes with I-block.

DETECTION METHOD We used CPD-Star system as our substrate for alkaline phosphatase. Expose film from 1 minute, 5 minute, 1 hour, up to overnight exposure but not seeing a signal at the correct size

POSITIVE AND NEGATIVE CONTROLS USED Positive control: nuclear extract of NRK cells. This nuclear extract has been verified that it contains nuclear components by probing with anti-Nups antibody. I do not have a negative control.

ANTIBODY STORAGE CONDITIONS 100ul at -20C

SAMPLE PREPARATION Nuclear extract in 10%sucrose, , 0.1mM MgCl2, 10mM TEA pH 7.4 and protease inhibitors (leupeptin, Pepstatin, TAME, BAME) Samples heated at 100C with sample loading buffer for 1minute before loading to 11% polyacrylamide gel

AMOUNT OF PROTEIN LOADED Serial dilution: 100ug, 50ug, 25ug and 12.5ug

ELECTROPHORESIS/GEL CONDITIONS 11% denaturing SDS-PAGE

TRANSFER AND BLOCKING CONDITIONS transfer overnight at 30V. blocking for 1 hour in I-block

SECONDARY ANTIBODY Anti-mouse antibody conjugated with alkaline phosphatase for 30 minutes at room temperature. 3 washes afterwards with I-block

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? Yes

WHAT STEPS HAVE YOU ALTERED? I increased the concentration of the antibody. Also increase the amount of proteins I loaded.

ANSWER:

I'm sorry to hear you are having a problem with ab8982.

This antibody has been purchased many times in the past few months without any reported problems, so I am fairly confident that we can modify your protocol and get the antibody to work for you. I would like to suggest the following modifications to your protocol: 1) Please incubate in primary antibody overnight at 4 degrees to allow for a slower more specific binding and a longer time for the antibody to interact with antigen, 2) Please block for 30 minutes at room temp and incubate in secondary at room temp for one hour, 3)I am not certain of the constituents of I-block, but we often use 5% milk to block and if you are not seeing a signal you can dilute the antibodies in TBS-T instead of blocking buffer, 4) We routinely use the ECL plus detected method as this is highly sensitive and specific. I have attached the protocol that I would use with the antibody below.

Western Blotting

1) To a sample of protein solution containing 1-100 ng of the target protein, add an equal volume of 2x SDS-PAGE sample buffer. For reduced samples, the sample buffer should be supplemented with DTT or 2-mercaptoethanol. For non-reduced samples, the DTT or 2-mercaptoethanol is not added (step 2 can also be left out dependent on sample). Denature the proteins by heating the sample to 95 oC, or boiling, for 5 min. 2)Load the sample onto an SDS-polyacrylamide gel and run the gel under standard conditions. 3) Transfer the proteins to a nitrocellulose or PVDF membrane using semi-dry or wet transfer methods. Please note: for PVDF it is essential to pre-wet the membrane in methanol prior to transfer. 4) If required, the efficiency of transfer can be determined by staining the membrane briefly (10 s) in Ponceau stain. The stain can be removed by washing in PBST or TBST. We would recommend not washing blots in distilled water as this can strip off proteins in some circumstances. 5) Block the membrane with a blocking buffer containing dried milk or BSA, or one from a commercial source (eg 5% w/v nonfat dry milk or 5% w/v BSA in PBST or TBST ). Incubate for 30 min at room temperature. 6)Dilute the primary antibody. We recommend incubating 1ml of primary antibody solution per 15 cm2 blot in a sealed bag, hybridisation tube or 50ml Falcon tubes can also be used (~2.5ml primary antibody/blot). Incubate overnight at 4 oC with agitation. 7) Rinse the blot in PBST or TBST and then wash twice for 5 min each and twice for 15 min at room temperature. 8)Dilute the horseradish peroxidase (HRP) labeled secondary antibody at the recommended dilution (1/5000 is usually a good working dilution although this needs to be optimised for the particular application). 9) Rinse the blot in PBST or TBST and then wash twice for 5 min each and twice for 15 min at room temperature. An optional PBS or TBS wash may be preformed as Tween may interfere with some ECL systems. 10) Perform ECL detection using the appropriate ECL solution. Please let me know if this helps and do not hesitate to contact us for further advice.

BATCH NUMBER 88913 ORDER NUMBER P0190B

DESCRIPTION OF THE PROBLEM No signal

SAMPLE nuclear extracts from cultured rat primary hepatocytes

PRIMARY ANTIBODY Lamin B1 antibody (ab8982) in TBST Incubated overnight (1:50 dilution) at 4 degrees C washed 3 times with TBST(20mM Tris, pH 7.5, 150mM Nacl, 0.1% Tween 20) for 10 mins each

SECONDARY ANTIBODY [a competitor] Anti-mouse raised in sheep diluted 1:5000 in TBST; incubated for 1 hr at room temperature washed 3 times with TBST; 10 mins each

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED Positive control: nuclear extract in which nuclear Nrf2 previously detected Negative control: cytosolic extracts from hepatocytes

protein bands verified on membrane using Ponceau S

ANTIBODY STORAGE CONDITIONS 50 ul aliquots stored at -20 degrees C.

SAMPLE PREPARATION Buffer used: 20mM HEPES, pH 7.9, 1.5mM MgCl2, 0.42M NaCl, 0.2mM EDTA, 25% (v/v) Glycerol. Protease Inhibitor Cocktail (Sigma) - 1:100 Phosphatase Inhibitor (Sigma)- 1:100 0.01M DTT Samples heated at 95 degrees C for 5 min

AMOUNT OF PROTEIN LOADED 40 ug

ELECTROPHORESIS/GEL CONDITIONS 10% Tris-HCl mini-gel (Bio-Rad)

TRANSFER AND BLOCKING CONDITIONS Transfer buffer: 25mM Tris, 192mM glycine, 20% methanol, 0.1% SDS Transferred for 2 hours

Blocking agent: 5% milk, 0.5% BSA

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? altered dilution of antibody- 1:30 to 1:200

ADDITIONAL NOTES absolutely no signal...blot clean....no background detected

ANSWER:

We have received information from the supplier of ab8982 apologising for this problem and currently re-testing the antibody and offering a replacement vial as soon as the new batch is quality approved.

I was unfortunately unable to find your order details to arrange the replacement vial, did you purchase the antibody via one of our distributors? If so please let me know and we will arrange the replacement via them. If you purchased directly with Abcam could you please provide me with the Abcam order number?

I look forward to hearing from you,

Thank you for your patience,

Follow-up to previous correspondance:

We diluted Lamin B1 antibody in 1XTBS-Tween with 5% milk and incubate overnight at 4 degrees. We have not tried unstripped blot with lamin B1, but with 1:50 dilution we quickly drained out the antibody. We loaded 50 micrograms of total proteins of the cell lysate. I do not know any positive control for this antibody, we just included a malignant breast cancer cell line and its siRNA knockouts of our genes in the western.

ANSWER:

Thank you very much for providing these further details. I would definitely recommend trying the antibody on a "fresh" membrane i.e. straight after transfer and blocking as the stripping can seriously damage proteins and prevent their detection by the primary antibody.

I look forward to hearing the results of this experiment,

I purchased a lamin B1 antibody (ab8982) from you. We used 1:50 dilution for western analysis of human breast cancer cell lines and saw no band at around 70 kDa (the molecular weight of lamin B1). We intended to use your antibody for a loading control, so we probed the membrane with one antibody and stripped it for lamin B1 western. To our surprise, the bands from the first antibody appeared again instead of the lamin B1 band. We are not happy with the performance of this antibody. Do you have any suggestion for a better loading control with molecular weight above 60kDa?

ANSWER:

I'm very sorry to hear you are having a problem with ab8982, it should work in WB. Could you please tell me how long you incubated the primary antibody for and in what buffer? Have you tried the antibody without stripping? What amount of protein do you load? What is the positive control?

I look forward to hearing from you and thank you for your patience,